Enzyme-free and amplification-free sequencing
1. A sequencing probe comprising a target binding domain and a barcode domain;
wherein the target binding domain comprises at least eight nucleotides and is capable of binding a target nucleic acid;
wherein the barcode domain comprises a synthetic backbone, the barcode domain comprising at least six attachment positions, each attachment position comprising at least one attachment region comprising at least one nucleic acid sequence capable of binding by a complementary nucleic acid molecule,
wherein each of the at least six attachment positions corresponds to one nucleotide in the target binding domain and each of the at least six attachment positions has a different nucleic acid sequence, an
Wherein the nucleic acid sequence of each of the at least six attachment positions determines the position and identity of a corresponding one of the target nucleic acids bound by the target binding domain.
2. The sequencing probe of claim 1, wherein the synthetic backbone comprises single-stranded DNA.
3. The sequencing probe of claim 1, wherein said sequencing probe comprises a double-stranded DNA spacer between said target binding domain and said barcode domain.
4. The sequencing probe of claim 1, wherein the number of nucleotides in a target binding domain is at least two more than the number of attachment regions in the barcode domain.
5. The sequencing probe of claim 1, wherein each position in the barcode domain has: (a) the same number of attachment regions; (b) an attachment region; or (c) more than one attachment region.
6. The sequencing probe of claim 1, wherein at least one complementary nucleic acid molecule comprises a detectable label.
7. The sequencing probe of claim 1, wherein at least one complementary nucleic acid molecule is directly linked to a primary nucleic acid molecule.
8. The sequencing probe of claim 1, wherein at least one complementary nucleic acid molecule is indirectly linked to a primary nucleic acid molecule via a nucleic acid spacer.
9. The sequencing probe of claim 1, wherein the at least one complementary nucleic acid comprises from about 8 nucleotides to about 20 nucleotides.
10. The sequencing probe of claim 1, wherein at least one complementary nucleic acid molecule comprises about 12 nucleotides.
11. The sequencing probe of claim 7 or claim 8, wherein at least one primary nucleic acid molecule is hybridized to at least one, two, three, four or five secondary nucleic acid molecules.
12. The sequencing probe of claim 11, wherein the one or more secondary nucleic acid molecules comprise at least one detectable label.
13. The sequencing probe of claim 11, wherein said at least one secondary nucleic acid molecule hybridizes to at least one, two, three, four, five, six, or seven tertiary nucleic acid molecules comprising at least one detectable label.
14. The sequencing probe of claim 1, wherein one or more attachment regions in a barcode domain are adjacent to at least one flanking single-stranded polynucleotide.
15. A population of sequencing probes comprising a plurality of sequencing probes of claim 1.
16. A method of nucleic acid sequencing comprising the steps of:
(1) hybridizing at least a first population of first sequencing probes comprising a plurality of sequencing probes of any of claims 1 to 14 to target nucleic acids immobilized to a substrate, wherein the target nucleic acids are immobilized to the substrate at one or more positions;
(2) binding a first complementary nucleic acid molecule comprising a detectable label or a first complementary nucleic acid molecule of a first reporter complex comprising a detectable label to a first attachment site of the at least six attachment sites;
(3) detecting the detectable label of the bound first complementary nucleic acid molecule or the detectable label of the bound first complementary nucleic acid molecule of the first reporter complex;
(4) identifying the position and identity of the first nucleotide in the immobilized target nucleic acid;
(5) binding the first attachment site to a first hybridizing nucleic acid molecule lacking a detectable label, thereby unbinding the first complementary nucleic acid molecule comprising the detectable label or the first complementary nucleic acid molecule of the first reporter complex comprising the detectable label;
(6) binding a second complementary nucleic acid molecule comprising a detectable label or a second complementary nucleic acid molecule comprising a second reporter complex of detectable labels to a second attachment site of the at least six attachment sites;
(7) detecting the detectable label of the bound second complementary nucleic acid molecule or the detectable label of the bound second complementary nucleic acid molecule of the second reporter complex;
(8) identifying the position and identity of a second nucleotide in the immobilized target nucleic acid;
(9) repeating steps (5) through (8) until each of the at least six attachment positions has been bound by a complementary nucleic acid molecule comprising a detectable label or a complementary nucleic acid molecule of a reporter complex comprising a detectable label and the detectable label of the bound complementary nucleic acid molecule or the detectable label of the bound complementary nucleic acid molecule of a reporter complex has been detected, thereby identifying a linear order of at least six nucleotides of at least a first region of the immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe; and
(10) at least one first population of first sequencing probes is de-hybridized from the nucleic acid.
17. The method of claim 16, wherein steps (5) and (6) occur sequentially or simultaneously.
18. The method of claim 16, wherein the first complementary nucleic acid molecule and the first hybridizing nucleic acid molecule lacking a detectable label comprise the same nucleic acid sequence.
19. The method of claim 16, wherein the first hybridizing nucleic acid molecule lacking a detectable label comprises a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the first attachment region.
20. The method of claim 16, further comprising
(11) Hybridizing at least one second population of second sequencing probes comprising a plurality of sequencing probes of any of claims 1 to 10 to target nucleic acids immobilized to a substrate, wherein the target nucleic acids are immobilized to the substrate at one or more positions, wherein the target binding domain of the first sequencing probe and the target binding domain of the second sequencing probe are different;
(12) binding a first complementary nucleic acid molecule comprising a detectable label or a first complementary nucleic acid molecule of a first reporter complex comprising a detectable label to a first attachment site of the at least six attachment sites;
(13) detecting the detectable label of the bound first complementary nucleic acid molecule or the detectable label of the bound first complementary nucleic acid molecule of the first reporter complex;
(14) identifying the position and identity of the first nucleotide in the immobilized target nucleic acid;
(15) binding a first hybridizing nucleic acid molecule lacking the detectable label to the first attachment site, thereby unbinding the first complementary nucleic acid molecule comprising the detectable label or the first complementary nucleic acid molecule of the first reporter complex comprising the detectable label;
(16) binding a second complementary nucleic acid molecule comprising a detectable label or a second complementary nucleic acid molecule comprising a second reporter complex of detectable labels to a second attachment site of the at least six attachment sites;
(17) detecting the detectable label of the bound second complementary nucleic acid molecule or the detectable label of the bound second complementary nucleic acid molecule of the second reporter complex;
(18) identifying the position and identity of a second nucleotide in the immobilized target nucleic acid;
(19) repeating steps (15) to (18) until each of the at least six attachment positions has been bound by a complementary nucleic acid molecule comprising a detectable label or a complementary nucleic acid molecule of a reporter complex comprising a detectable label and the detectable label of the bound complementary nucleic acid molecule or the detectable label of the bound complementary nucleic acid molecule of a reporter complex has been detected, thereby identifying a linear order of at least six nucleotides of at least a second region of the immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe; and
(20) (ii) dehybridizing at least one second population of the second sequencing probes to the immobilized target nucleic acid.
21. The method of claim 20, further comprising the step of assembling each identified linear order of nucleotides of at least a first region and at least a second region of the immobilized target nucleic acid, thereby identifying the sequence of the immobilized target nucleic acid.
22. Kit comprising a substrate, a plurality of sequencing probes according to any of claims 1 to 14, at least two capture probes, wherein the capture probes comprise a nucleic acid sequence hybridizing to a region of the target nucleic acid different from the region of the target nucleic acid hybridizing to the sequencing probes,
at least six complementary nucleic acid molecules comprising a detectable label, wherein the complementary nucleic acid comprising the detectable label comprises a nucleic acid sequence that hybridizes to one of the sequencing probe and one of the at least six attachment positions of the detectable label,
at least six complementary nucleic acid molecules lacking a detectable label, wherein the complementary nucleic acid lacking a detectable label comprises a nucleic acid sequence that hybridizes to one of the at least six attachment positions of the sequencing probe, and instructions for use.
23. A kit comprising the consumable sequencing card used in the method of claim 16.
Background
Various methods of nucleic acid sequencing exist, i.e., methods of determining the precise order of nucleotides within a nucleic acid molecule. Current methods require enzymatic (e.g., PCR and/or by cloning) amplification of nucleic acids. Further enzymatic polymerization is required to generate a detectable signal by a light detection means. Such amplification and polymerization steps are expensive and/or time consuming. Thus, there is a need in the art for amplification-free and enzyme-free methods of nucleic acid sequencing. The present invention addresses these needs.
Summary of The Invention
The present invention provides sequencing probes, methods, kits and devices that provide enzyme-free, amplification-free and library-free nucleic acid sequencing with long read lengths and low error rates. In addition, the methods, kits and devices have rapid sample-to-answer (sample-to-answer) capabilities. These features are particularly useful for sequencing in a clinical setting.
Provided herein are sequencing probes comprising a target binding domain and a barcode domain. The target binding domain and barcode domain may be operably linked, e.g., covalently linked. The sequencing probe optionally comprises a spacer between the target binding domain and the barcode domain. The spacer may be any polymer with suitable mechanical properties, such as a single-stranded or double-stranded DNA spacer (of 1 to 100 nucleotides, for example 2 to 50 nucleotides). Non-limiting examples of double stranded DNA spacers include the sequences covered by SEQ ID NO 25 to SEQ ID NO 29.
The target binding domain comprises at least four nucleotides (e.g., 4, 5,6, 7, 8,9, 10, 11, 12 or more) and is capable of binding a target nucleic acid (e.g., DNA, RNA, and PNA). The barcode domain comprises a synthetic backbone having at least a first position comprising one or more attachment regions. The barcode domain may have 1,2, 3, 4, 5,6, 7, 8,9, 10, 11, 12 or more positions; each location has one or more (e.g., one to fifty) attachment regions; each attachment region comprises at least one (i.e., one to fifty, e.g., ten to thirty copies of a nucleic acid sequence) capable of reversibly binding to a complementary nucleic acid molecule (RNA or DNA). Some locations in the barcode domain may have more attachment areas than others; optionally, each location in the barcode domain has the same number of attachment regions. The nucleic acid sequence of the first attachment region determines the position and identity of a first nucleotide in the target nucleic acid bound by a first nucleotide of the target binding domain, while the nucleic acid sequence of the second attachment region determines the position and identity of a second nucleotide in the target nucleic acid bound by a second nucleotide of the target binding domain. Likewise, the nucleic acid sequence of the sixth attachment region determines the position and identity of the sixth nucleotide in the target nucleic acid that is bound by the sixth nucleotide of the target binding domain. In embodiments, the synthetic backbone comprises a polysaccharide, a polynucleotide (e.g., single or double stranded DNA or RNA), a peptide nucleic acid, or a polypeptide. The number of nucleotides in the target binding domain is equal to or greater than the number of positions in the barcode domain (e.g., 1,2, 3, 4, or more). Each attachment region in a particular position of a barcode domain may comprise one copy of the same nucleic acid sequence and/or multiple copies of the same nucleic acid sequence. However, the attachment region will include a nucleic acid sequence that is different from the attachment region at a different position of the barcode domain, even when both attachment regions identify the same type of nucleotide (e.g., adenine, thymine, cytosine, guanine, uracil, and analogs thereof). The attachment region may be linked to a modified monomer, such as a modified nucleotide, in the synthetic backbone, thereby creating a branch with respect to the backbone. The attachment region may be part of a polynucleotide sequence of the synthetic backbone. The one or more attachment regions may be adjacent to at least one flanking single-stranded polynucleotide, that is, the attachment region may be operably linked to a 5 'flanking single-stranded polynucleotide and/or a 3' flanking single-stranded polynucleotide. The attachment region with or without one or both flanking single-stranded polynucleotides may hybridize to a hybridizing nucleic acid molecule lacking a detectable label. The hybrid nucleic acid molecule lacking the detectable label may be between about 4 to about 20 nucleotides in length, for example 12 nucleotides or longer.
The attachment region may be bound by a complementary nucleic acid comprising a detectable label. Each complementary nucleic acid may comprise a detectable label.
Alternatively, the attachment region may be bound by a complementary nucleic acid that is part of the reporter complex (comprising the detectable label). Complementary nucleic acids (comprising a detectable label or reporter complex) can be between about 4 to about 20 nucleotides in length, e.g., about 8, 10, 12, and 14 nucleotides or more. In the reporter complex, the complementary nucleic acid is linked (directly or indirectly) to a primary nucleic acid molecule. The complementary nucleic acid can be indirectly linked to the primary nucleic acid molecule via a single-or double-stranded nucleic acid linker (e.g., a polynucleotide comprising 1 to 100 nucleotides). The primary nucleic acid hybridizes to one or more (e.g., 1,2, 3, 4, 5,6, 7, 8,9, 10, or more) secondary nucleic acids. Each secondary nucleic acid hybridizes to one or more (e.g., 1,2, 3, 4, 5,6, 7, 8,9, 10, or more) tertiary nucleic acids; the tertiary nucleic acid comprises one or more detectable labels. The or each secondary nucleic acid may comprise a region which does not hybridise to a primary nucleic acid molecule and does not hybridise to a tertiary nucleic acid molecule ("extra handle"); the region may be four or more (e.g., about 6 to about 40, such as about 8, 10, 12, and 14) nucleotides in length. The region that does not hybridize to a primary nucleic acid molecule and does not hybridize to a tertiary nucleic acid molecule may comprise the nucleotide sequence of a complementary nucleic acid molecule linked to the primary nucleic acid molecule. This region may be located at the end of the second nucleic acid which hybridizes to the primary nucleic acid distal to its end. By having an "extra handle" of nucleotide sequences comprising complementary nucleic acids, the probability and speed of binding of the reporter complex to the sequencing probe is greatly increased. In any embodiment or aspect of the invention, when the reporter complex comprises an "additional handle", the reporter complex may hybridize to the sequencing probe via the complementary nucleic acid of the reporter complex or via the "additional handle". Thus, for example, the phrase "first complementary nucleic acid molecule that binds to the first attachment region.
In embodiments, the terms "barcode domain" and "synthetic backbone" are synonymous.
Provided herein are methods for sequencing nucleic acids using the sequencing probes of the invention. The method comprises the following steps: (1) hybridizing at least one sequencing probe of the invention to a target nucleic acid immobilized to a substrate (e.g., at 1,2, 3, 4, 5,6, 7, 8,9, 10 or more positions); (2) binding a first complementary nucleic acid molecule (RNA or DNA) having a detectable label (e.g., a fluorescent label) or a first complementary nucleic acid molecule of a first reporter complex comprising a detectable label (e.g., a fluorescent label) to the first attachment region; (3) detecting the detectable label, and (4) identifying the position and identity of the first nucleotide in the immobilized target nucleic acid. Optionally, the immobilized target nucleic acid is elongated before being bound by the probe. The method further comprises the steps of: (5) contacting the first attachment region (with or without one or both flanking single-stranded polynucleotides) with a first hybridizing nucleic acid molecule lacking a detectable label, thereby not binding to the first complementary nucleic acid molecule with the detectable label or the first complementary nucleic acid molecule comprising the first reporter complex of the detectable label and binding the first hybridizing nucleic acid molecule lacking the detectable label to at least the first attachment region; (6) binding a second complementary nucleic acid molecule with a detectable label or a complementary nucleic acid molecule of a second reporter complex comprising a detectable label to the second attachment region; (7) detecting the detectable label; and (8) identifying the position and identity of the second nucleotide in the immobilized target nucleic acid. Repeating steps (5) to (8) until each nucleotide in the immobilized target nucleic acid corresponding to the target binding domain has been identified. Steps (5) and (6) may occur simultaneously or sequentially. Each (e.g., first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or higher) complementary nucleic acid molecule (with a portion of a detectable label or reporter complex) has the same nucleic acid sequence as its corresponding (i.e., first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or higher) hybridizing nucleic acid molecule lacking a detectable label. Immobilizing the target nucleic acid to the substrate by binding the first and/or second position of the target nucleic acid with the first and/or second capture probe; each capture probe comprises an affinity tag that selectively binds to a substrate. The first and/or second location can be at or near the end of the target nucleic acid. The substrate may be any solid support known in the art, such as coated slides and microfluidic devices (e.g., coated with streptavidin). Other locations distal to the end of the target nucleic acid can be selectively bound to the substrate. Nucleic acids can be elongated by applying a force sufficient to extend the target nucleic acid (e.g., gravity, hydrodynamic forces, electromagnetic forces, flow stretching, receding meniscus technique (and combinations thereof)).
Provided herein are methods for sequencing nucleic acids using one population of probes of the invention or a plurality of populations of probes of the invention. The method comprises the following steps: (1) hybridizing a first population of sequencing probes (of the invention) to the target nucleic acid immobilized to the substrate (wherein each sequencing probe in the first population is unhybridized to the immobilized target nucleic acid under about the same conditions (e.g., level of chaotropic agent, temperature, salt concentration, pH, and hydrodynamic force); (2) binding a plurality of first complementary nucleic acid molecules each having a detectable label or a plurality of first complementary nucleic acid molecules of a plurality of first reporter complexes each complex comprising a detectable label to a first attachment region in each sequencing probe in the first population; (3) detecting the detectable label; (4) identifying the position and identity of a plurality of first nucleotides in the immobilized target nucleic acid hybridized by the sequencing probes in the first population; (5) contacting each first attachment region of each sequencing probe of the first population with a plurality of first hybridizing nucleic acid molecules lacking a detectable label, thereby not binding a first complementary nucleic acid molecule having a detectable label or a reporter complex, and binding the first hybridizing nucleic acid molecules lacking a detectable label to each first attachment region; (6) binding a plurality of second complementary nucleic acid molecules each having a detectable label or a plurality of second complementary nucleic acid molecules of a plurality of second reporter complexes each complex comprising a detectable label to a second attachment region in each sequencing probe in the first population; (7) detecting the detectable label; and (8) identifying the position and identity of a plurality of second nucleotides in the immobilized target nucleic acid hybridized by the sequencing probes in the first population. In step (9), steps (5) through (8) are repeated until each nucleotide in the immobilized target nucleic acid corresponding to the target binding domain of each sequencing probe in the first population has been identified. Steps (5) and (6) may occur simultaneously or sequentially. Thus, a linear order of nucleotides is identified for the region of immobilized target nucleic acid to which the target binding domains of the sequencing probes in the first population of sequencing probes hybridize.
In embodiments, when multiple populations (i.e., more than one population) of probes are used, the method further comprises the steps of: (10) de-hybridizing each sequencing probe of the first population from the nucleic acid; (11) removing each de-hybridized sequencing probe of the first population; (12) hybridizing at least a second population of sequencing probes of the invention, wherein each sequencing probe in the second population dehybridizes to the immobilized target nucleic acid under about the same conditions and dehybridizes to the immobilized target nucleic acid under conditions different from the sequencing probes in the first population; (13) binding a plurality of first complementary nucleic acid molecules each having a detectable label or a plurality of first complementary nucleic acid molecules of a plurality of first reporter complexes each complex comprising a detectable label to the first attachment region in each sequencing probe in the second population; (14) detecting the detectable label; (15) identifying the location and identity of a plurality of first nucleotides in the immobilized target nucleic acid hybridized by the sequencing probes in the second population; (16) contacting each first attachment region of each sequencing probe of the second population with a plurality of first hybridizing nucleic acid molecules lacking the detectable label, thereby unbinding the first complementary nucleic acid molecule (with the detectable label or from the reporter complex), and binding the first hybridizing nucleic acid molecule lacking the detectable label to each first attachment region; (17) binding a plurality of second complementary nucleic acid molecules each having a detectable label or a plurality of second complementary nucleic acid molecules of a plurality of second reporter complexes each complex comprising a detectable label to a second attachment region in each sequencing probe in the second population; (18) detecting the detectable label; (19) identifying the location and identity of a plurality of second nucleotides in the immobilized target nucleic acid hybridized by the sequencing probes in the second population; and (20) repeating steps (16) through (19) until a linear order of nucleotides has been identified for the region of immobilized target nucleic acid hybridized by the target binding domains of the sequencing probes in the second population of sequencing probes. Steps (16) and (17) may occur simultaneously or sequentially.
Each sequencing probe in the second population can be de-hybridized from the immobilized target nucleic acid under different conditions (e.g., higher temperature, higher level of chaotropic agent, higher salt concentration, higher flow rate, and different pH) than the average conditions under which the sequencing probes in the first population de-hybridize from the target nucleic acid.
However, when more than two populations of probes are used, then the probes in the two consecutive populations can hybridize under different conditions, and the probes in the non-consecutive populations can hybridize under similar conditions. As an example, probes in the first population and the third population may hybridize under similar conditions. In embodiments, a contiguous population of probes is hybridized under increasingly more stringent conditions (e.g., higher chaotropic agent levels, salt concentrations, and temperatures). For microfluidic devices, using temperature as an example, the first population of probes may remain hybridized at a first temperature, but proceed to hybridize at a second temperature that is higher than the first temperature. The second population of probes can remain hybridized at the second temperature, but remain hybridized at a third temperature that is greater than the second temperature. In this example, the temperature of the solution flowing through the target nucleic acid for the initial probe population (containing the reagents required for the present method) is lower than the temperature of the solution flowing through the target nucleic acid for the subsequent probe population.
In some embodiments, after the probe population has been used, the probe population is de-hybridized from the target nucleic acid, and a new aliquot of the same probe population is used. For example, subsequent aliquots of the first probe population are hybridized after the first probe population has been hybridized, detected, and dehybridized. Alternatively, as an example, a first population of probes may be de-hybridized and replaced with a second population of probes; once the second population has been detected and de-hybridized, a subsequent aliquot of the first probe population is hybridized to the target nucleic acid. Thus, probes in a subsequent population can hybridize to regions of a target nucleic acid that have been previously sequenced (thereby obtaining repeated and/or validated sequence information) or probes in a subsequent population can hybridize to regions of a target nucleic acid that have not been previously sequenced (thereby obtaining new sequence information). Thus, the probe population may be re-aliquoted when the previous reads are unsatisfactory (for any reason) and/or improve the accuracy of the alignment resulting from the sequencing read.
Probes that hybridize and de-hybridize under similar conditions can have similar lengths of their target binding domains, GC contents, or frequencies of repeated bases, and combinations thereof. The relationship between Tm and length of oligonucleotides is taught, for example, by Sugimoto et al,Biochemistry, 34, 11211-6。
when more than two populations of probes are used, the steps as described for the first and second populations of sequencing probes are repeated with additional populations of probes (e.g., 10 to 100 to 1000 populations). The number of probe populations used will depend on a variety of factors including, but not limited to, the size of the target nucleic acid, the number of unique probes in each population, the degree of overlap between sequencing probes desired, and the enrichment of the probes into the target region.
The probe population can contain additional sequencing probes directed to a particular region of interest in the target nucleic acid, such as a region containing a mutation (e.g., a point mutation) or SNP allele. The probe population may contain fewer sequencing probes directed to particular regions of less interest in the target nucleic acid.
The population of sequencing probes can be compartmentalized into a discrete smaller collection of sequencing probes. Compartmentalization can be based on the predicted melting temperature of the target binding domain in the sequencing probe and/or the sequence motif of the target binding domain in the sequencing probe. Compartmentalization may be based on empirically derived rules. Different sets of sequencing probes can be reacted with the target nucleic acid using different reaction conditions, e.g., based on temperature, salt concentration, and/or buffer content. Compartmentalization can be performed to cover target nucleic acids with uniform coverage. Compartmentalization can be performed to cover target nucleic acids with known coverage profiles.
The length of the target binding domain in the sequencing probe population can be shortened to increase the coverage of probes in a particular region of the target nucleic acid. The length of the target binding domain in the sequencing probe population can be increased to reduce coverage of the probe in a particular region of the target nucleic acid, e.g., above the resolution limit of the sequencing apparatus.
Alternatively or additionally, the concentration of sequencing probes in the population can be increased to increase coverage of probes in a particular region of the target nucleic acid. The concentration of sequencing probes can be reduced to reduce the coverage of probes in a particular region of the target nucleic acid, e.g., above the resolution limit of the sequencing apparatus.
The method for sequencing a nucleic acid further comprises the step of assembling each identified linear order of nucleotides of each region of the immobilized target nucleic acid, thereby identifying the sequence of the immobilized target nucleic acid. The step of assembling uses a non-transitory computer readable storage medium having an executable program stored thereon that instructs a microprocessor to arrange each identified linear order of nucleotides to obtain a sequence of the nucleic acid. Assembly may occur in "real-time," i.e., while data is being collected from the sequencing probes, rather than after all data has been collected.
The target nucleic acid, i.e., the sequenced target nucleic acid, can be between about 4 and 1,000,000 nucleotides in length. The target may comprise the entire, intact chromosome or a length thereof that is greater than 1,000,000 nucleotides in length.
An apparatus for carrying out the method of the invention is provided herein.
Provided herein are kits comprising the sequencing probes of the invention and for performing the methods of the invention. In embodiments, the kit comprises a substrate capable of immobilizing nucleic acids via capture probes, a plurality of sequencing probes of the invention, at least one capture probe, at least one complementary nucleic acid moiety having a detectable labelAt least one complementary nucleic acid molecule lacking a detectable label, and instructions for use. In embodiments, the kit comprises about or at least 4096 unique sequencing probes. 4096 is the minimum number of unique probes (i.e., for probes with six attachment regions in the barcode domain) necessary to include each possible hexamer combination. Here, "4096" is achieved because there are four nucleotide options for the six positions: 46. For a probe set with four attachment regions in the barcode domain, only 256 would be required (i.e., 4)4Species) unique probes. For a probe set with 8 nucleotides in its target binding domain, 4 would be required8Species (i.e., 65,536) unique probes. For a probe set with 10 nucleotides in its target binding domain, 4 would be required10Species (i.e., 1,048,576 species) unique probes.
In embodiments, the kit comprises about or at least twenty four different complementary nucleic acid molecules having a detectable label, and about or at least twenty four different hybridizing nucleic acid molecules lacking a detectable label. As a non-limiting example, a complementary nucleic acid can bind to an attachment region having a sequence of one of SEQ ID NOs 1 to 24. Additional exemplary sequences that can be included in the barcode domain are set forth in SEQ ID NO: 42 through SEQ ID NO: 81. In practice, the nucleotide sequence is not limited; preferably, it lacks substantial homology to known nucleotide sequences (e.g., 50% to 99.9%); this helps to avoid undesired hybridization of the complementary nucleic acid and the target nucleic acid.
Any of the above aspects and embodiments may be combined with any other aspect or embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the specification, the singular forms also include the plural forms unless the context clearly dictates otherwise; by way of example, the terms "a", "an" and "the" are to be construed as being singular or plural, and the term "or" is to be construed as being inclusive. By way of example, "an element" means one or more elements. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the indicated value. Unless otherwise clear from the context, all numbers provided herein are modified by the term "about".
Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Citation of references herein is not an admission as to prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Brief Description of Drawings
This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the patent and trademark office upon request and payment of the necessary fee.
The above and further features will be more clearly understood from the detailed description when taken in conjunction with the accompanying drawings.
Fig. 1-5 show schematic diagrams of exemplary sequencing probes of the invention.
FIGS. 6A to 6D are schematic diagrams showing variants of the sequencing probe of the present invention.
FIG. 7 shows a schematic of the target binding domain of a sequencing probe of the invention; the domain comprises zero, two or four nucleotides having a universal base.
FIGS. 8A to 8E illustrate the steps of the sequencing method of the present invention.
FIG. 9A shows the initial steps of the sequencing method of the invention.
Figure 9B shows a schematic of a reporter complex comprising a detectable label.
Fig. 9C shows a plurality of reporter complexes each comprising a detectable label.
FIGS. 9D to 9G show further steps of the sequencing method starting in FIG. 9A.
FIG. 10 shows an alternative illustration of the steps shown in FIGS. 9D and 9E, and exemplary data obtained thereby. The fragment of the sequencing probe has the sequence of SEQ ID NO: 82.
Fig. 11 illustrates a variation of the method shown in fig. 10. The fragment of the sequencing probe shown likewise has the sequence of SEQ ID NO: 82.
FIG. 12 illustrates the method of the present invention.
FIG. 13 compares the steps required in the sequencing method of the invention with those required in other sequencing methods.
Fig. 14 and 15 illustrate performance measurements that can be obtained by the present invention.
FIG. 16 compares the sequencing rates, reads, and clinical utility of the present invention with various other sequencing methods/devices.
FIG. 17 shows the low raw error rate of the sequencing method of the invention. The template sequence has the sequence of SEQ ID NO 83.
FIG. 18 compares sequencing data obtainable from the present invention with other sequencing methods.
FIG. 19 shows the single base specificity of the sequencing method of the invention. The template and probe sequences shown (from top to bottom) have the sequences SEQ ID NO: 84 to SEQ ID NO: 88.
FIG. 20A shows various designs of reporter complexes of the invention.
Figure 20B shows fluorescence counts obtained from the reporter complexes shown in figure 20A.
Fig. 20C shows an exemplary formulation for constructing a reporting complex of the present invention.
Fig. 21A shows the design of a reporting composite comprising an "extra handle".
Figure 21B shows fluorescence counts obtained from reporter complexes with "extra handle".
Fig. 22A and 22B show hybridization kinetics for two exemplary designs of reporter complexes of the invention.
FIG. 23 shows a schematic diagram of a sequencing probe of the invention used in a method different from that shown in FIGS. 8-12.
FIG. 24 shows a schematic of a consumable sequencing card that can be used in the present invention.
FIG. 25 shows the mismatch detection of the 10-mer as described in example 3. The nucleotides shown (from top to bottom) have the sequences SEQ ID NO 89 to SEQ ID NO 99.
FIG. 26 shows the ability to hybridize according to the size of the target binding domain described in example 3. Due to the very high reporter concentration, the background was high and no prior purification was performed. The nucleotides shown (from top to bottom) have the sequence SEQ ID NO 100 to SEQ ID NO 104.
Figure 27 shows a comparison between a single spot compared to the full length reporter. The results of the single spots show a hybridization rate 1000-fold higher than that of the full-length barcode (condition 100nM target, 30 min hybridization).
Detailed Description
The present invention provides sequencing probes, methods, kits and devices that provide enzyme-free, amplification-free and library-free nucleic acid sequencing with long read lengths and low error rates.
Sequencing probe
The present invention relates to sequencing probes comprising a target binding domain and a barcode domain. Non-limiting examples of sequencing probes of the invention are shown in figures 1 to 6.
FIG. 1 shows a schematic representation of a sequencing probe of the invention. The exemplary sequencing probe has a target binding domain of six nucleotides, each of which corresponds to a position in a barcode domain (which contains one or more attachment regions). Note the first attachment region; which corresponds to the nucleotide of the target nucleic acid that is bound by the first nucleotide in the target binding domain. Note the third position on the barcode domain. Note the fifth location containing two attachment regions. Each location on the barcode domain may have multiple attachment regions. For example, a location may have 1 to 50 attachment regions. Some locations in the barcode domain may have more attachment areas than others (as shown here for location 5 relative to locations 1-4 and 6); optionally, each location in a barcode domain has the same number of attachment regions (see, e.g., fig. 2,3,5, and 6). Although not shown, each attachment region comprises a nucleic acid sequence capable of reversibly binding to at least one (i.e., one to fifty, e.g., ten to thirty) copies of a complementary nucleic acid molecule (RNA or DNA). In fig. 1, the attachment region is essential for the linear polynucleotide molecules that make up the barcode domain.
FIG. 2 shows a schematic of a sequencing probe of the invention. The exemplary sequencing probe has a target binding domain of six nucleotides, each of which corresponds to an attachment region in a barcode domain. Note the first attachment region; which corresponds to the nucleotide of the target nucleic acid that is bound by the first nucleotide in the target binding domain. A fourth position of the barcode domain is circled, which includes a portion of the barcode domain and two fourth attachment regions. Note the two sixth attachment areas. Here, each location has two attachment areas; however, each location on the barcode domain may have one attachment region or multiple attachment regions, for example 2 to 50 attachment regions. Although not shown, each attachment region comprises a nucleic acid sequence capable of reversibly binding to at least one (i.e., one to fifty, e.g., ten to thirty) copies of a complementary nucleic acid molecule (RNA or DNA). In fig. 2, the barcode domain is a linear polynucleotide molecule that connects the attachment regions; the attachment region is not essential to the polynucleotide molecule.
FIG. 3 shows another schematic of a sequencing probe of the invention. The exemplary sequencing probe has a target binding domain of four nucleotides, where the four nucleotides correspond to four positions in the barcode domain. Each location shows an attachment area with three connections.
FIG. 4 shows another schematic of a sequencing probe of the invention. The exemplary sequencing probe has a target binding domain of ten nucleotides. However, only the first six nucleotides correspond to six positions in the barcode domain. Adding the seventh to tenth nucleotides (from "n1To n4"indicate) to increaseThe length of the target binding domain, thereby affecting the likelihood that the probe will hybridize and remain hybridized to the target nucleic acid. In embodiments, an "n" nucleotide may precede the nucleotide corresponding to a position in the barcode domain. In embodiments, an "n" nucleotide may follow a nucleotide corresponding to a position in a barcode domain. In FIG. 4, four "n" nucleotides are shown; however, the target binding domain may comprise more than four "n" nucleotides. An "n" nucleotide can have a universal base (e.g., inosine, 2' -deoxyinosine (hypoxanthine deoxynucleotide) derivatives, nitroindoles, nitroindazole analogs, and hydrophobic aromatic non-hydrogen bonding groups) that can base pair with any of the four typical bases.
Another sequencing probe of the invention is shown in FIG. 5. Here, "n" nucleotides precede and follow the nucleotides corresponding to positions in the barcode domain. The exemplary sequencing probe shown has a target binding domain of ten nucleotides. However, only three to eight nucleotides in the target binding domain correspond to six positions (first to sixth) in the barcode domain. Adding the first, second, ninth and tenth nucleotides (consisting of "n1To n4"indicated") to increase the length of the target binding domain. In FIG. 5, four "n" nucleotides are shown; however, the target binding domain may include more or less than four "n" nucleotides.
FIGS. 6A-6D show variations of the sequencing probe of FIG. 1. In fig. 6A, the linear order of nucleotides in the target binding domain and the linear order of the attachment regions in the barcode domain proceeds from left to right (relative to the diagram). In FIG. 6B, the linear order of the nucleotides in the target binding domain and the linear order of the attachment regions in the barcode domain proceeds from right to left (relative to the diagram). In fig. 6C, the linear order of the nucleotides in the target binding domain is inverted relative to the linear order of the attachment regions in the barcode domain. In any probe of the invention, the nucleotides in the target binding domain and the attachment regions in the barcode domain lack strict order as long as the probe is designed such that each nucleotide in the target binding domain corresponds to one or more attachment domains in the target binding domain; the lack of strict order is shown in FIG. 6D. Any of the probes of the invention (e.g., those exemplified in figures 1 to 5) can have the order of nucleotides and attachment regions as shown in figure 6.
The target binding domain has at least four nucleotides, for example at least 4, 5,6, 7, 8,9, 10, 11, 12 or more nucleotides. The target binding domain is preferably a polynucleotide. The target binding domain is capable of binding a target nucleic acid.
The probe may include multiple copies of the target binding domain operably linked to a synthetic backbone.
Probes can be designed to control the likelihood of hybridization and/or de-hybridization and the rate at which these occur. Generally, the lower the Tm of a probe, the faster and more likely it will be to de-hybridize with/from a target nucleic acid. Thus, using a lower Tm probe will reduce the number of probes that bind to the target nucleic acid.
The length of the target binding domain affects in part the probability that the probe will hybridize to and remain hybridized to the target nucleic acid. Generally, the longer the target binding domain (greater number of nucleotides), the less likely that a complementary sequence will be present in the target nucleotide. Conversely, the shorter the target binding domain, the greater the likelihood that a complementary sequence will be present in the target nucleotide. For example, the probability that a tetramer sequence will be located in a target nucleic acid is 1/256, compared to 1/4096. Thus, a collection of shorter probes will likely bind at more positions for a given nucleic acid segment when compared to a collection of longer probes.
Figure 7 shows a 10 mer target binding domain. In some embodiments, the target binding domain comprises four universal bases (identified as "U") base-paired to any one of four canonical nucleotides (A, G, C and T)b"). In embodiments, the target binding domain comprises one to six (e.g., 2 and 4) universal bases. The target binding domain may not include universal nucleotides. FIG. 7 indicates that a "complete" population of probes with 6 specific nucleotides in the target binding domain would require 4096 unique probes, and a "complete" population of probes with 10 specific nucleotides would require about 100-ten thousand unique probes.
In some cases, probes with shorter target binding domains are preferred to increase the read-out in a given nucleic acid segment, thereby enriching the coverage of the target nucleic acid or a portion of the target nucleic acid, in particular a portion of particular interest, for example when detecting a mutation or a SNP allele.
However, it may be preferred to have a smaller number of probes bound to the target nucleic acid, since too many probes in the presence region may cause their detectable labels to overlap, thereby preventing resolution of two adjacent probes. This is explained as follows. Considering a one nucleotide length of 0.34nm and considering a lateral (x-y) spatial resolution of the sequencing device of about 200nm, the resolution limit of the sequencing device is about 588 base pairs (i.e. 1 nucleotide/0.34 nm x 200 nm). That is, the sequencing device described above will not be able to resolve signals from two probes hybridized to a target nucleic acid when the two probes are within about 588 base pairs of each other. Thus, depending on the resolution of the sequencing device, two probes will need to be spaced apart by about 600bp before their detectable labels can be resolved into distinct "spots". Therefore, at optimal spacing, there should be a single probe for every 600bp target nucleic acid. Various software methods (e.g., using fluorescence intensity values and wavelength-dependent ratios) can be used to monitor, limit, and potentially deconvolute the number of probes hybridized within a resolvable region of a target nucleic acid and design a population of probes accordingly. In addition, detectable labels (e.g., fluorescent labels) that provide a more discrete signal may be selected. Furthermore, methods in the literature (e.g., Small and Parthasarhy: "Superresolution localization methods"Annu. Rev. Phys Chem2014; 65:107-25) describe structure illumination and various super-resolution methods that reduce the resolution limit of sequencing microscopes up to 10's of nanometers. The use of higher resolution sequencing devices allows the use of probes with shorter target binding domains.
As mentioned above, the Tm of the designed probe can affect the number of probes that hybridize to the target nucleic acid. Alternatively or additionally, the concentration of sequencing probes in the population can be increased to increase coverage of probes in a particular region of the target nucleic acid. The concentration of sequencing probes can be reduced to reduce the coverage of probes in a particular region of the target nucleic acid, e.g., above the resolution limit of the sequencing apparatus.
The term "target nucleic acid" shall mean a nucleic acid molecule (DNA, RNA or PNA) whose sequence is to be determined by the probes, methods and devices of the invention. In general, the terms "target nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleic acid fragment," "oligonucleotide," and "polynucleotide" are used interchangeably and are intended to include, but are not limited to, polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides or analogs thereof) that can be of various lengths. Non-limiting examples of nucleic acids include genes, gene fragments, exons, introns, intergenic DNA (including but not limited to heterochromosomal DNA), messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozymes, small interfering RNA (sirna), non-coding RNA (ncrna), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of sequence, isolated RNA of sequence, nucleic acid probes, and primers.
The present method directly sequences nucleic acid molecules obtained from a sample (e.g., a sample from an organism), preferably without a transformation (or amplification) step. As an example, for RNA-based sequencing, the present method does not require the conversion of an RNA molecule to a DNA molecule (i.e., via cDNA synthesis) before the sequence is available. Since no amplification or transformation is required, nucleic acids sequenced in the present invention will retain any unique bases and/or epigenetic markers present in the nucleic acid when the nucleic acid is in a sample or when it is obtained from a sample. Such unique bases and/or epigenetic markers are lost in sequencing methods known in the art.
The target nucleic acid can be obtained from any sample or source of nucleic acid, such as any cell, tissue, or organism, in vitro, chemical synthesizer, and the like. The target nucleic acid can be obtained by any art-recognized method. In embodiments, the nucleic acid is obtained from a blood sample of a clinical subject. Nucleic acids can be extracted, isolated or purified from a source or sample using methods and reagents well known in the art.
Nucleic acid molecules comprising a target nucleic acid can be fragmented by any means known in the art. Preferably, the fragmentation is carried out enzymatically or mechanically. The mechanical means may be sonication or physical shearing. Enzymatic means can be performed by digestion with a nuclease (e.g., deoxyribonuclease I (DNase I)) or one or more restriction endonucleases.
When the nucleic acid molecule comprising the target nucleic acid is an intact chromosome, steps should be taken to avoid fragmenting the chromosome.
Target nucleic acids can include natural or non-natural nucleotides, including modified nucleotides, as is well known in the art.
Probes of the invention can have a total length (including the target binding domain, barcode domain, and any optional domain) of about 20 nanometers to about 50 nanometers. The backbone of the probe may be a polynucleotide molecule comprising about 120 nucleotides.
The barcode domain comprises a synthetic backbone. The synthetic backbone and the target binding domain are operably linked, e.g., covalently attached or attached via a linker. The synthetic backbone may comprise any material, such as a polysaccharide, a polynucleotide, a polymer, a plastic, a fiber, a peptide nucleic acid, or a polypeptide. Preferably, the synthetic backbone is rigid. In embodiments, the backbone comprises a "DNA origami" of six DNA duplexes (see, e.g., Lin et al, "submicroscale geotrichally encoded fluorescent barcodes self-assembled from DNA.Nature Chemistry2012 Oct, 4(10) 832-9). The bar code can be made of DNA origami tiles (Jungmann et al), “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT”, Nature Methods, Vol. 11, No. 3, 2014)。
The barcode domain comprises a plurality of positions, for example one, two, three, four, five, six, seven, eight, nine, ten or more positions. The number of positions can be less than, equal to, or greater than the number of nucleotides in the target binding domain. It is preferred to include additional nucleotides in the target binding domain compared to the number of positions in the backbone domain, for example one, two, three, four, five, six, seven, eight, nine, ten or more nucleotides. The length of the barcode domain is not limited as long as there is sufficient space for at least four locations as described above.
Each position in the barcode domain corresponds to a nucleotide in the target binding domain and thus corresponds to a nucleotide in the target nucleic acid. As an example, the first position in the barcode domain corresponds to the first nucleotide in the target binding domain and the sixth position in the barcode domain corresponds to the sixth nucleotide in the target binding domain.
Each location in the barcode domain contains at least one attachment region, for example, one to fifty or more attachment regions. Some locations in a barcode domain may have more attachment regions than others (e.g., a first location may have three attachment regions, while a second location may have two attachment locations); optionally, each location in the barcode domain has the same number of attachment regions. Each attachment region comprises at least one (i.e., one to fifty, e.g., ten to thirty) copies) of a nucleic acid sequence capable of being reversibly bound by a complementary nucleic acid molecule (e.g., DNA or RNA). In an example, the nucleic acid sequence in the first attachment region determines the position and identity of a first nucleotide in the target nucleic acid that is bound by the first nucleotide of the target binding domain. Each attachment region may be linked to a modified monomer (e.g., a modified nucleotide) in the synthetic backbone such that the attachment region branches from the synthetic backbone. In embodiments, the attachment region is essential to the polynucleotide backbone; that is, the backbone is a single polynucleotide and the attachment regions are part of a single polynucleotide sequence. In embodiments, the terms "barcode domain" and "synthetic backbone" are synonymous.
The nucleic acid sequence in the attachment region determines the position and identity of the nucleotide in the target nucleic acid that is bound by the nucleotide of the target binding domain of the sequencing probe. In a probe, each attachment region will have a unique overall sequence. In fact, each position on the barcode domain may have an attachment region comprising a nucleic acid sequence encoding one of the four nucleotides, i.e. specific for one of adenine, thymine/uracil, cytosine and guanine. In addition, an attachment region at a first position (and e.g., encoding a cytosine) will include a nucleic acid sequence that is different from an attachment region at a second position (and e.g., encoding a cytosine). Thus, for a nucleic acid sequence in the attachment region encoding the first position of thymine, there will be no binding of a complementary nucleic acid molecule that identifies adenine in the target nucleic acid corresponding to the first nucleotide of the target binding domain. Furthermore, for the attachment region at the second position, there will be no binding of the complementary nucleic acid molecule that identifies adenine in the target nucleic acid corresponding to the first nucleotide of the target binding domain.
Each location on the barcode field may comprise one or more (at most fifty, preferably ten to thirty) attachment regions; thus, each attachment region may bind one or more (up to fifty, preferably ten to thirty) complementary nucleic acid molecules. As an example, the probe in fig. 1 has a fifth position with two attachment regions, and the probe in fig. 2 has a second position with six attachment regions. In embodiments, the nucleic acid sequences of the attachment regions at the positions are identical; thus, the complementary nucleic acid molecules that bind those attachment regions are identical. In alternative embodiments, the nucleic acid sequences of the attachment regions of the locations are different; thus, the complementary nucleic acid molecules that bind those attachment regions are different, e.g., each comprises a different nucleic acid sequence and/or a detectable label. Thus, in an alternative embodiment, the combination of different nucleic acid molecules (e.g., their detectable labels) attached to the attachment region provides a code for identifying the nucleotide in the target nucleic acid.
For illustrative purposes only, table 1 provides exemplary sequences for the accessory regions of sequencing probes having up to six positions in their barcode domain and a detectable label on the complementary nucleic acid to which they bind.
Table 1:
nucleotides/stripes in target binding domainsPosition in the shape code domain
Nucleotide, its preparation and use
Nucleic acid sequences in the attachment region (5 'to 3')
Detectable labels for complementary nucleic acids
SEQ ID NO
1
A
ATACATCTAG
GFP
1
1
G
GATCTACATA
RFP
2
1
C
TTAGGTAAAG
CFP
3
1
U/T
TCTTCATTAC
YFP
4
2
A
ATGAATCTAC
GFP
5
2
G
TCAATGTATG
RFP
6
2
C
AATTGAGTAC
CFP
7
2
U/T
ATGTTAATGG
YFP
8
3
A
AATTAGGATG
GFP
9
3
G
ATAATGGATC
RFP
10
3
C
TAATAAGGTG
CFP
11
3
U/T
TAGTTAGAGC
YFP
12
4
A
ATAGAGAAGG
GFP
13
4
G
TTGATGATAC
RFP
14
4
C
ATAGTGATTC
CFP
15
4
U/T
TATAACGATG
YFP
16
5
A
TTAAGTTTAG
GFP
17
5
G
ATACGTTATG
RFP
18
5
C
TGTACTATAG
CFP
19
5
U/T
TTAACAAGTG
YFP
20
6
A
AACTATGTAC
GFP
21
6
G
TAACTATGAC
RFP
22
6
C
ACTAATGTTC
CFP
23
6
U/T
TCATTGAATG
YFP
24
As seen in Table 1, the nucleic acid sequence of the first attachment region may be one of SEQ ID NO 1 to SEQ ID NO 4 and the nucleic acid sequence of the second attachment may be one of SEQ ID NO 5 to SEQ ID NO 8. When the first nucleotide in the target nucleic acid is adenine, the nucleic acid sequence of the first attachment region will have the sequence of SEQ ID NO. 1, and when the second nucleotide in the target nucleic acid is adenine, the nucleic acid sequence of the second attachment region will have the sequence of SEQ ID NO. 5.
In embodiments, the complementary nucleic acid molecule may be bound by a detectable label. In an alternative embodiment, the complementary nucleic acid is bound to a reporter complex comprising a detectable label.
The nucleotide sequence of the complementary nucleic acid is not limited; preferably, it lacks substantial homology to known nucleotide sequences (e.g., 50% to 99.9%); this helps to avoid undesired hybridization of the complementary nucleic acid and the target nucleic acid.
An example of a reporter complex useful in the present invention is shown in fig. 9B. In this example, the complementary nucleic acid is linked to a primary nucleic acid molecule, which in turn hybridizes to a plurality of secondary nucleic acid molecules, each of which in turn hybridizes to a plurality of tertiary nucleic acid molecules having one or more detectable labels attached thereto.
In embodiments, the primary nucleic acid molecule may comprise about 90 nucleotides. The secondary nucleic acid molecule may comprise about 87 nucleotides. The tertiary nucleic acid molecule may comprise about 15 nucleotides.
Fig. 9C shows a population of exemplary reporter complexes. Included in the upper left panel of fig. 9C are four complexes that hybridize to attachment region 1 of the probe. For each possible nucleotide at nucleotide position 1 of the target binding domain of the probe, there is one type of reporter complex present. Here, if position 1 of the reporter domain of the probe is bound by a reporter complex with a "blue" detectable label while performing the sequence method of the invention, the first nucleotide in the target binding domain is identified as adenine. Alternatively, if position 1 is bound by a reporter complex having a "green" detectable label, the first nucleotide in the target binding domain is identified as thymine.
The reporter complex can have various designs. For example, a primary nucleic acid molecule can hybridize to at least one (e.g., 1,2, 3, 4, 5,6, 7, 8,9, 10, or more) secondary nucleic acid molecule. Each secondary nucleic acid molecule can hybridize to at least one (e.g., 1,2, 3, 4, 5,6, 7, 8,9, 10, or more) tertiary nucleic acid molecule. An exemplary reporter complex is shown in fig. 20A. Here, a "4 x 3" reporter complex has one primary nucleic acid molecule (which is linked to a complementary nucleic acid molecule) hybridized to four secondary nucleic acid molecules, each hybridized to three trinucleotide molecules (each comprising a detectable label). In this figure, each complementary nucleic acid of the complex is 12 nucleotides in length ("12 bases"); however, the length of the complementary nucleic acid is not limited, and may be less than 12 or more than 12 nucleotides. The bottom right complex includes a spacer between its complementary nucleic acid and its primary nucleic acid molecule. The spacer is identified as being 20 to 40 nucleotides long; however, the length of the spacer is not limiting, and it may be shorter than 20 nucleotides or longer than 40 nucleotides.
Figure 20B shows the variable mean (fluorescence) counts obtained from the four exemplary reporter complexes shown in figure 20A. In FIG. 20B, 10pM of biotinylated target template was attached to a streptavidin-coated flow cell surface, 10nM of reporter complex was flowed onto the flow cell; after 1 minute of incubation, the flow cell was washed, imaged, and the fluorescence signature was counted.
In embodiments, the reporter complex is "pre-constructed". That is, each polynucleotide in the complex is hybridized prior to contacting the complex with the probe. An exemplary recipe for the pre-construction of five exemplary reporting complexes is shown in fig. 20C.
Figure 21A shows an alternative reporter complex in which the secondary nucleic acid molecule has an "extra handle" that is not hybridized to the tertiary nucleic acid molecule and is distal to the primary nucleic acid molecule. In this figure, each "extra handle" is 12 nucleotides long ("12 mer"); however, their length is not limited and may be less than 12 or more than 12 nucleotides. In embodiments, the "additional handles" each comprise a nucleotide sequence of a complementary nucleic acid; thus, when the reporter complex comprises an "additional handle", the reporter complex can hybridize to the sequencing probe via the complementary nucleic acid of the reporter complex or via the "additional handle". Thus, the probability of binding of the reporter complex to the sequencing probe is increased. The "extra handle" design may also improve hybridization kinetics. Without being bound by theory, the "additional handle" substantially increases the effective concentration of complementary nucleic acids of the reporter complex.
Fig. 21B shows the variable mean (fluorescence) counts obtained from five exemplary reporter complexes with "extra handle" using the procedure described for fig. 20B.
Fig. 22A and 22B show hybridization kinetics and fluorescence intensity for two exemplary reporter complexes. By about 5 minutes, the total count began to plateau, indicating that most of the reporter complex added had found a useful target.
The detectable moiety, label, or reporter can be bound to the complementary nucleic acid or tertiary nucleic acid molecule in a variety of ways, including direct or indirect attachment of a detectable moiety, such as a fluorescent moiety, a colorimetric moiety, or the like. The person skilled in the art can refer to references for marker nucleic acids. Examples of fluorescent moieties include, but are not limited to, Yellow Fluorescent Protein (YFP), Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Red Fluorescent Protein (RFP), umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanine, dansyl chloride, phycocyanin, phycoerythrin, and the like. Fluorescent labels and their attachment to nucleotides and/or Oligonucleotides are described in a number of Reviews, including Haughland, Handbook of Fluorescent Probes and Research Chemicals, ninth edition (Molecular Probes, Inc., Eugene, 2002), Keller and Manak, DNA Probes, 2nd edition (Stockton Press, New York, 1993), Eckstein, editors of oligonucleotide and Oligonucleotides, A Practical apparatus (IRL Press, Oxford, 1991), and Wetmur, clinical Reviews in Biochemistry and Molecular Biology, 26: 227-. Specific methods suitable for use in the present invention are disclosed in the following sample of references: U.S. patent nos. 4,757,141; 5,151,507, respectively; and 5,091,519. In one aspect, one or more fluorescent dyes are used as labels for labeled target sequences, e.g., as disclosed by U.S. Pat. No. 5,188,934(4, 7-dichlorofluorescein dye); 5,366,860 (spectrally resolvable rhodamine dyes); 5,847,162(4, 7-dichlororhodamine dye); 4,318,846 (ether substituted fluorescein dyes); 5,800,996 (energy transfer dyes); lee et al 5,066,580 (xanthine dyes); 5,688,648 (energy transfer dyes); and the like. Labeling can also be performed with quantum dots, as disclosed in the following patents and patent publications: U.S. Pat. nos. 6,322,901; 6,576,291, respectively; 6,423,551; 6,251,303; 6,319,426, respectively; 6,426,513, respectively; 6,444,143, respectively; 5,990,479; 6,207,392; 2002/0045045, respectively; and 2003/0017264. As used herein, the term "fluorescent label" comprises a signaling moiety that conveys information by the fluorescent absorption and/or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectral characteristics, energy transfer, and the like.
Commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and/or oligonucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED-5-dUTP, CASCADE BLUE-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMTR-14-dUTP, RHOAMINE GREEN 488-5-dUTP, OREGON GREENR 488-5-dUTP, TEXAS RED-12-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-ALdUTP, BODIPO-5-dUTP, BODIPOR-532-5-dUTP, BODIPY TMR-14-dUTP, and BODIPY TMR-5-dUTP, ALEXA FLUOR 568-5-dUTP, ALEXA FLUOR 594-5-dUTP, ALEXA FLUOR 546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED ™ 5-UTP, mCherry, CASCADE BLUE-7-UTP, BODIPY TM FL-14-UTP, BODIPY TMR-14-UTP, BODIPY TM TR-14-UTP, RHODINE GREEN-5-UTP, ALEXA FLUOR 488-5-UTP, LEXA FLUOR 546-14-UTP (Molecular Probes, Inc. Eugene, OR), and the like. Alternatively, the above fluorophores and those mentioned herein may be added during oligonucleotide synthesis using, for example, phosphoramidite (phosphoroamidate) or NHS chemistry. Protocols for the custom synthesis of nucleotides with other fluorophores are known in the art (see, Henegariu et al (2000) Nature Biotechnol. 18: 345). 2-amino purines are fluorescent bases that can be incorporated directly into oligonucleotide sequences during their synthesis. Nucleic acids can also be first stained with intercalating dyes such as DAPI, YOYO-1, ethidium bromide, cyanine dyes (e.g., SYBR Green), and the like.
Other fluorophores that may be used for post-synthetic attachment include, but are not limited to, ALEXA FLUOR [ 350 ], ALEXA FLUOR [ 405 ], ALEXA FLUOR [ 430 ], ALEXA FLUOR [ 532 ], ALEXA FLUOR [ 546 ], ALEXA FLUOR [ 568 ], ALEXA FLUOR [ 594 ], ALEXA FLUOR [ 647 ], BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY CasDIY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, BocaBlue, Dacayrallow, NSYl, Marelin B, Maracina, Oregon 488, Oregen [ 514 ], Oregan [ 514 ], Blue [ 82 ], rhodamine B, Cyanene [ 82 ], rhodamine ] dye, Roche, Cyanene [ 3 ], rhodamine, Roche, Cyben [ 2 ] Cy5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), and the like. FRET tandem fluorophores can also be used, including but not limited to PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, and the like.
Metallic silver or gold particles may be used to enhance the signal from fluorescently labeled nucleotide and/or oligonucleotide sequences (Lakowicz et al (2003) BioTechniques 34: 62).
Other suitable labels for oligonucleotide sequences may include fluorescein (FAM, FITC), digoxigenin, Dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6XHis), phospho-amino acids (e.g., P-tyr, P-ser, P-thr), and the like. In one embodiment, the following hapten/antibody pairs are used for detection, wherein each antibody is derivatized with a detectable label: biotin/alpha-biotin, digoxigenin/a-digoxigenin, Dinitrophenol (DNP)/a-DNP, 5-carboxyfluorescein (FAM)/a-FAM.
The detectable labels described herein are spectrally resolvable. Reference to "spectrally resolvable" of a plurality of fluorescent labels means that the fluorescence emission bands of the labels are sufficiently different, i.e., sufficiently non-overlapping, that the molecular tags attached to the respective labels can be distinguished based on the fluorescent signals generated by the respective labels by standard light detection systems, e.g., systems employing band-pass filters and photomultiplier tubes, etc., as in U.S. patent No. 4,230,558; 4,811,218; and the like, or by the system described in Wheeless et al, pages 21-76, Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York, 1985). In one aspect, spectrally resolvable organic dyes, such as fluorescein, rhodamine, and the like, mean that the wavelength emission maxima are spaced apart by at least 20nm, and on the other hand are spaced apart by at least 40 nm. In another aspect, spectrally resolvable chelated lanthanide compounds, quantum dots, and the like mean that the wavelength emission maxima are spaced apart by at least 10nm, and in another aspect, by at least 15 nm.
Sequencing method
The present invention relates to methods for sequencing nucleic acids using the sequencing probes of the invention. Examples of such methods are shown in fig. 8 to 12.
The method comprises reversibly hybridizing at least one sequencing probe of the invention with a target nucleic acid immobilized to a substrate (e.g., at 1,2, 3, 4, 5,6, 7, 8,9, 10 or more positions).
The substrate can be any solid support known in the art, such as coated slides and microfluidic devices, which are capable of immobilizing a target nucleic acid. In certain embodiments, the substrate is a surface, a membrane, a bead, a porous material, an electrode, or an array. The target nucleic acid may be immobilized on any substrate apparent to those skilled in the art.
In embodiments, the target nucleic acid is bound by a capture probe comprising a domain complementary to a portion of the target nucleic acid. The moiety may be an end of the target nucleic acid, or may not be towards the end.
Exemplary useful matrices include matrices comprising binding moieties selected from ligands, antigens, carbohydrates, nucleic acids, receptors, lectins, and antibodies. The capture probe comprises a binding moiety capable of binding to a binding moiety of a substrate. Exemplary useful substrates comprising reactive moieties include, but are not limited to, surfaces comprising epoxy, aldehyde, gold, hydrazide, thiol, NHS-ester, amine, thiol, carboxylate, maleimide, hydroxymethylphosphine, imide, isocyanate, hydroxyl, pentafluorophenyl ester, psoralen, pyridyl disulfide or vinyl sulfone, polyethylene glycol (PEG), hydrogel or mixtures thereof. Such surfaces may be obtained from commercial sources or prepared according to standard techniques. Exemplary useful matrices containing reactive moieties include, but are not limited to, the OptAlay-DNA NHS group (Accler8), Nexterion Slide AL (Schott), and Nexterion Slide E (Schott).
In embodiments, the binding moiety of the capture probe is biotin and the matrix comprises avidin (e.g., streptavidin). Useful matrices containing avidin are commercially available, including TB0200 (Accelr8), SAD6, SAD20, SAD100, SAD500, SAD2000 (Xantec), SuperAvidin (Array-It), streptavidin slides (catalog # MPC 000, Xenopore), and STREPTAVIDIDINnslide (catalog #439003, Greiner Bio-one).
In embodiments, the binding moiety of the capture probe is avidin (e.g., streptavidin) and the matrix comprises biotin. Useful substrates comprising biotin that are commercially available include, but are not limited to, Optiarray-biotin (Accler8), BD6, BD20, BD100, BD500, and BD2000 (Xantec).
In embodiments, the binding moiety of the capture probe may comprise a reactive moiety capable of binding to the substrate by photoactivation. The matrix may comprise a photoreactive moiety, or the first portion of the nanotransmitter may comprise a photoreactive moiety. Some examples of photoreactive moieties include aryl azides, such as N ((2-pyridyldithio) ethyl) -4-azidosalicylamide; fluorinated aryl azides such as 4-azido-2, 3,5, 6-tetrafluorobenzoic acid; benzophenone-based agents such as succinimidyl 4-benzoylbenzoic acid esters; and 5-bromo-deoxyuridine.
In embodiments, the binding moiety of the capture probe may be immobilized to the substrate via other binding pairs apparent to those skilled in the art.
After binding to the substrate, the nucleic acids can be elongated by applying a force sufficient to extend the target nucleic acids (e.g., gravity, hydrodynamic forces, electromagnetic force "electrostretching," flow stretching, receding meniscus techniques, and combinations thereof).
The target nucleic acid can be bound by a second capture probe comprising a domain complementary to a second portion of the target nucleic acid. The moiety may be an end of the target nucleic acid, or may not be towards the end. Binding of the second capture probe can occur after or during extension of the target nucleic acid, or the second capture probe can bind to an unextended target nucleic acid. The second capture probe may have binding as described above.
The capture probe may comprise or be associated with a detectable label (i.e., a fiducial).
The capture probe is capable of isolating a target nucleic acid from a sample. Here, a capture probe is added to a sample comprising the target nucleic acid. The capture probe binds to the target nucleic acid via a region of the capture probe that is complementary to a region of the target nucleic acid. When the target nucleic acid contacts a substrate comprising a moiety that binds to the binding moiety of the capture probe, the nucleic acid is immobilized to the substrate.
To ensure that the user "captures" as many target nucleic acid molecules as possible from a highly fragmented sample, it is helpful to include multiple capture probes, each complementary to a different region of the target nucleic acid. For example, there can be three sets of capture probes, where a first set is complementary to a region of the target nucleic acid near its 5 'end, a second set is complementary to a region in the middle of the target nucleic acid, and a third set is near its 3' end. This can be generalized to "n regions of interest" for each target nucleic acid. In this example, each individual collection of fragmented target nucleic acids is bound to a capture probe that comprises or binds a biotin tag. 1/n of the input sample (where n = number of different regions in the target nucleic acid) is separated for each collection chamber. The capture probe binds to the target nucleic acid of interest. The target nucleic acid is then immobilized to the avidin molecule attached to the substrate via the biotin of the capture probe. Optionally, the target nucleic acid is stretched, e.g., via flow or electrostatic force. All n sets can be stretched and bound simultaneously, or to maximize the number of fully stretched molecules, set 1 (which captures up to the 5' region) can be stretched and bound first; the assembly 2 (which captures the middle of the target region) can then be stretched and bound; finally, the set 3 may be stretched and bonded.
The number of different capture probes required is inversely proportional to the size of the target nucleic acid fragment. In other words, for highly fragmented target nucleic acids, more capture probes will be required. For sample types with highly fragmented and degraded target nucleic acids (e.g., formalin-fixed paraffin-embedded tissue), it may be useful to include multiple capture probe sets. On the other hand, for samples with long target nucleic acid fragments, such as isolated nucleic acids obtained in vitro, a single capture probe at the 5' end may be sufficient.
The region of the target nucleic acid between two capture probes or after one capture probe and before the end of the target nucleic acid is referred to herein as a "gap". The gap is a portion of the target nucleic acid that is available for binding by the sequencing probes of the invention. The smallest gap is the target binding domain length (e.g., 4 to 10 nucleotides), and the largest gap is the majority of the entire chromosome.
The immobilized target nucleic acid is shown in FIG. 12. Here, the two capture probes are identified as a "5 'capture probe" and a "3' capture probe".
FIG. 8A shows a schematic of a sequencing probe bound to a target nucleic acid. Here, the target nucleic acid has thymidine (T). A first set of complementary nucleic acids comprising detectable labels or reporter complexes is shown at the top, each member of the set having a different detectable label (e.g., thymidine identified by a green signal) and a different nucleotide sequence. The first nucleotide in the target binding domain binds to a T in the target nucleic acid. The first attachment region of the probe includes one or more nucleotide sequences that indicate that the first nucleotide in the target binding domain of the probe binds a thymidine. Thus, only the complementary nucleic acid of thymidine binds to the first position of the barcode domain. As shown, a first thymine-encoding complementary nucleic acid comprising a detectable label or a reporter complex comprising a detectable label is bound to the attachment region at the first position of the barcode domain of the probe.
The number of sets of complementary nucleic acids or reporter complexes is the same as the number of positions in the barcode domain. Thus, for a barcode domain with six positions, six sets will be cycled through on the probe.
Alternatively, the probe may be hybridized at its first position to a complementary nucleic acid or reporter complex comprising a detectable label prior to contacting the target nucleic acid with the probe. Thus, a probe is capable of emitting a detectable signal from its first position when contacted with its target nucleic acid, and does not have to provide a first set of complementary nucleic acids or reporter complexes directed to the first position on the barcode domain.
FIG. 8B continues the method shown in FIG. 8A. Here, the first complementary nucleic acid (or reporter complex) of thymidine that binds to the attachment region at the first position of the barcode domain has been replaced with a first hybridizing nucleic acid that is for thymidine and lacks a detectable label. The first hybridizing nucleic acid for thymidine and lacking the detectable label replaces the previously bound complementary nucleic acid or previously bound reporter complex comprising the detectable label. Thus, position 1 of the barcode domain no longer emits a detectable signal.
In embodiments, a complementary nucleic acid or reporter complex comprising a detectable label may be removed from the attachment region, but not replaced with a hybridizing nucleic acid lacking the detectable label. This may be done, for example, by adding chaotropic agents, increasing the temperature, changing the salt concentration, adjusting the pH and/or applying fluid power. In these embodiments, fewer reagents (i.e., hybridized nucleic acids lacking a detectable label) are required.
Fig. 8C continues the method of the claimed invention. Here, the target nucleic acid has cytidine (C) and thymidine (T) preceding it. A second set of complementary nucleic acids or reporter complexes is shown at the top, each member of the set having a different detectable label and a different nucleotide sequence. Furthermore, the nucleotide sequences of the complementary nucleic acids of the first set or of the complementary nucleic acids of the reporter complex are different from the nucleotide sequences of those of the second set. However, the base-specific detectable label is common to the collection of complementary nucleic acids, e.g., thymidine is identified by a green signal. Here, the second nucleotide in the target binding domain binds to a C in the target nucleic acid. The second attachment region of the probe has a nucleotide sequence that indicates that the second nucleotide in the target binding domain of the probe binds cytidine. Thus, only complementary nucleic acids or reporter complexes from the second repertoire and for cytidine comprising detectable labels bind to the second position of the barcode domain. As shown, a second complementary nucleic acid encoding cytidine, or a reporter complex, binds at a second position in the barcode domain of the probe.
In an embodiment, the step shown in fig. 8C follows the step shown in fig. 8B. Here, once the first set of complementary nucleic acids or reporter complexes (of fig. 8A) has been replaced with the first hybridizing nucleic acids (in fig. 8B) lacking a detectable label, a second set of complementary nucleic acids or reporter complexes is provided (as shown in fig. 8C). Alternatively, the steps shown in fig. 8C are performed simultaneously with the steps shown in fig. 8B. Here, a first hybridized nucleic acid lacking a detectable label (as shown in fig. 8B) is provided concurrently with a second set of complementary nucleic acids or reporter complexes (as shown in fig. 8C).
Fig. 8D continues the method shown in fig. 8C. Here, the first to fifth positions on the barcode domain are bound by a complementary nucleic acid or reporter complex comprising a detectable label and have been replaced by a hybridizing nucleic acid lacking the detectable label. The sixth position of the barcode domain is now bound by a complementary nucleic acid or reporter complex comprising a detectable label, which identifies the sixth position in the target binding domain as binding to guanine (G).
As mentioned above, a complementary nucleic acid or reporter complex comprising a detectable label may be removed from the attachment region, but not replaced with a hybridizing nucleic acid lacking the detectable label.
If desired, the exchange rate of the detectable label can be accelerated by incorporating a small single-stranded oligonucleotide that accelerates the exchange rate of the detectable label(e.g., "Toe-Hold" Probes; see, e.g., Seeling et al, “Catalyzed Relaxation of a Metastable DNA Fuel”; J. Am. Chem. Soc. 2006, 128(37), pp12211-12220)。
The complementary nucleic acid or reporter complex can be replaced at the final position of the barcode domain (sixth position in fig. 8D); however, this may not be necessary when the sequencing probe is replaced with another sequencing probe. Indeed, the sequencing probe of FIG. 8D can now be de-hybridized and removed from the target nucleic acid and replaced with a second (overlapping or non-overlapping) sequencing probe that has not been bound by any complementary nucleic acid, as shown in FIG. 8E. The probes in fig. 8E may be included in the second probe population.
Fig. 9A and 9D to 9G show the method steps of the invention, like fig. 8A to 8E; however, fig. 9A and 9D to 9G clearly show that the reporter complex (comprising the detectable label) binds to the attachment region of the sequencing probe. FIGS. 9D and 9E show the fluorescent signal emitted by the probe hybridized to the reporter complex. FIGS. 9D and 9E show that the target nucleic acid has a sequence of "T-A".
Fig. 10 summarizes the steps shown in fig. 9D and 9E. The nucleotide sequences of the exemplary probes are shown at the top of the figure and identify the important domains of the probes. The probe comprises an optional double stranded DNA spacer between its target binding domain and its barcode domain. The barcode domain includes, in order, a "side 1" portion, an "AR-1/side 2" portion, an "AR-2" portion, and an "AR-2/side 3" portion. In step 1, "AR-1 detection" hybridizes to the "AR-1" and "AR-1/flanking 2" portions of the probe. An "AR-1 detection" corresponds to a reporter complex encoding a first position thymidine or a complementary nucleic acid comprising a detectable label. Thus, step 1 corresponds to fig. 9D. In step 2, "lacking 1" hybridizes to the "flanking 1" and "AR-1" portions of the probe. "lack of 1" corresponds to a hybridized nucleic acid specific for the first attachment region of the probe that lacks the detectable label (shown as a black bar covering the first attachment region in fig. 9E). By hybridizing at the "flanking 1" position 5' to the reporter complex or complementary nucleic acid, the hybridizing nucleic acid more effectively displaces the reporter complex/complementary nucleic acid from the probe. The "flanking" portions are also referred to as "Toe-Holds". In step 3, "AR-2 detection" hybridizes to the "AR-2" and "AR-2/flanking 3" portions of the probe. The "AR-2 detection" corresponds to a reporter complex or a complementary nucleic acid encoding a guanine at the second position comprising a detectable label. Thus, step 3 corresponds to fig. 9E. In this embodiment, a hybridizing nucleic acid lacking the detectable label and a complementary nucleic acid/reporter complex comprising the detectable label are provided sequentially.
Optionally, a hybridizing nucleic acid lacking the detectable label and a complementary nucleic acid/reporter complex comprising the detectable label are provided simultaneously. This alternative embodiment is shown in fig. 11. In step 2, "lacking 1" (hybridized nucleic acid lacking detectable label) is provided along with "AR-2 detection" (reporter complex encoding guanine at the second position). This alternative embodiment may be more efficient than the embodiment shown in fig. 10, as it combines two steps into one.
FIG. 12 illustrates the method of the present invention. Here, the target nucleic acid is captured and immobilized at two locations, thereby creating a "gap" to which the probe can bind. The first probe population is hybridized to the target nucleic acid and the detectable label is detected. Repeat the initial steps with the second probe population, the third probe population, to more than 100 probe populations. The use of about 100 probe populations provides about 5-fold coverage of each nucleotide in the target nucleic acid. Fig. 12 provides an estimated rate of reading time based on the time required to detect a signal from one field of view (FOV).
The distribution of probes along the length of the target nucleic acid is critical for the resolution of detectable signals. As discussed above, the resolution limit of two detectable labels is about 600 nucleotides. Preferably, each sequencing probe in the probe population will bind no closer than 600 nucleotides to each other. As discussed above, 600 nucleotides is the resolution limit of a typical sequencing device. In this case, the sequencing probe will provide a single read; this is shown in fig. 12 at the leftmost resolution limit point.
Randomly, but depending in part on the length of the target binding domain, the Tm of the probe, and the concentration of the probe used, two different sequencing probes in a population can bind within 600 nucleotides of each other. In this case, the unordered multiple reads would emanate from a single resolution limit; this is shown in fig. 12 at the second resolution limit.
Alternatively or additionally, the concentration of sequencing probes in the population can be reduced to reduce the coverage of probes in a particular region of the target nucleic acid, e.g., above the resolution limit of the sequencing apparatus, thereby producing a single read from the resolution limit point.
Figure 23 shows a schematic of a sequencing probe different from that used in figures 8 to 12. Here, each position on the barcode domain is bound by a complementary nucleic acid or reporter complex comprising a detectable label. Thus, in this example, six nucleotide sequences can be read without the need for sequential replacement of complementary nucleic acids. The use of such sequencing probes will reduce the time to obtain sequence information, since many steps of the method are omitted. However, the probe would benefit from non-overlapping detectable labels, e.g., fluorophores excited by or emitting light at non-overlapping wavelengths.
The method further comprises the step of assembling each identified linear order of nucleotides of each region of the immobilized target nucleic acid, thereby identifying the sequence of the immobilized target nucleic acid. The step of assembling uses a non-transitory computer readable storage medium having an executable program stored thereon. The program instructs the microprocessor to arrange each identified linear order of nucleotides for each region of the target nucleic acid to obtain a sequence of the nucleic acid. Assembly may occur in "real-time," i.e., while data is being collected from the sequencing probes, rather than after all data has been collected.
Any of the above aspects and embodiments may be combined with any other aspect or embodiment disclosed herein in the summary and/or detailed description section.
Defining:
in certain exemplary embodiments, the terms "annealing" and "hybridizing" as used herein are used interchangeably to denote the formation of a stable duplex. In one aspect, a stable duplex means that the duplex structure is not disrupted by stringent washing under conditions such as: a temperature of about 5 ℃ below or about 5 ℃ above the Tm of the strands of the duplex and a low monovalent salt concentration, e.g., less than 0.2M, or less than 0.1M or a salt concentration known to those skilled in the art. The term "perfectly matched" when used with reference to a duplex means that the polynucleotide and/or oligonucleotide strands making up the duplex form a double stranded structure with each other such that each nucleotide in each strand undergoes Watson-Crick base pairing with a nucleotide in the other strand. The term "duplex" includes, but is not limited to, pairs of nucleoside analogs such as deoxyinosine, nucleosides with 2-aminopurine bases, PNA, and the like, which may be employed. A "mismatch" in a duplex between two oligonucleotides means that a pair of nucleotides in the duplex cannot undergo Watson-Crick bonding.
As used herein, the term "hybridization conditions" will generally include salt concentrations of less than about 1M, more typically less than about 500mM, and even more typically less than about 200 mM. Hybridization temperatures can be as low as 5 ℃, but are typically greater than 22 ℃, more typically greater than about 30 ℃, and often greater than about 37 ℃. Hybridization is typically performed under stringent conditions, e.g., conditions under which the probe will specifically hybridize to its target sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. Since other factors can affect the stringency of hybridization, including the base composition and length of the complementary strands, the presence of organic solvents and the degree of base mismatching, the combination of parameters is more important than the absolute measurement of either alone.
Typically, stringent conditions are selected so as to be about 5 ℃ lower than the Tm for the particular sequence at a defined ionic strength and pH. Exemplary stringent conditions include a salt concentration of at least 0.01M to no more than 1M Na ion concentration (or other salt) at a pH of 7.0 to 8.3 and a temperature of at least 25 ℃. For example, conditions of 5 XSSPE (750mM NaCl, 50mM sodium phosphate, 5mM EDTA, pH7.4) and a temperature of 25-30 ℃ are suitable for allele-specific probe hybridization. For stringent conditions, see, for example, Sambrook, Fritsche and Maniatis, "Molecular Cloning A Laboratory Manual, 2nd Ed." Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1 st edition, BIOS Scientific Publishers Limited (1999). As used herein, the term "specifically hybridizes to …" or "specifically hybridizes to …" or similar terms means that the molecule substantially binds, duplexes, or hybridizes under stringent conditions to one or more specific nucleotide sequences.
A detectable label associated with a particular location of a probe may be "read" (e.g., its detected fluorescence) one or more times; "reading" may be synonymous with the term "base call". Multiple reads improve accuracy. A target nucleic acid sequence is "read" when a contiguous sequence segment of sequence information derived from a single initial target molecule is detected; typically, this is produced via a multichannel consensus (as defined below). As used herein, the term "coverage" or "depth of coverage" refers to the number of times a target region has been sequenced (via discrete reads) and aligned with a reference sequence. Read coverage is the total reads mapped to a particular reference target sequence; base coverage is the total number of base calls made at a particular genomic position.
As used herein, the "hybe and seq cycle" refers to all steps required to detect each attachment region on a particular probe or population of probes. For example, for a probe capable of detecting six positions on a target nucleic acid, one "hybe and seq cycle" would include at least hybridizing the probe to the target nucleic acid, hybridizing a complementary nucleic acid/reporter complex to the attachment region of each of the six positions on the barcode domain of the probe, and detecting a detectable label associated with each of the six positions.
The term "k-mer probe" is synonymous with the probe of the present invention.
When aligning two or more sequences from discrete reads, the overlapping portions can be combined to produce a single consensus sequence. These bases are common at positions where the overlapping portions have the same base (single aligned column). Various rules can be used to generate a consensus of the locations where there is an inconsistency between overlapping sequences. Simple majority rules use the most common bases in the column as consensus. A "multichannel consensus" is an alignment of all discrete probe readings from a single target molecule. Each base position within a single target molecule can be interrogated with different levels of redundancy or overlap depending on the total number of cycles of probe population/vote applied; in general, redundancy increases the confidence level of base calls.
"Primary accuracy" is a measure of the system's inherent ability to correctly identify a base. The original accuracy depends on the sequencing technique. "common precision" is the ability of the system to correctly identify bases using additional reads and statistical power. "specificity" refers to the percentage of reads that map to the intended target per run of total reads. "homogeneity" refers to the variability of sequence coverage of a target region; high uniformity is associated with low variability. This feature is typically reported as the fraction of the targeted area covered by ≧ 20% of the average depth of coverage of all targeted areas. Random errors (i.e., inherent sequencing chemistry errors) can be easily corrected with "multi-pass" sequencing of the same target nucleic acid; given a sufficient number of passes, essentially "perfect consensus" or "error-free" sequencing can be achieved.
The methods described herein may be implemented and/or recorded using any device capable of implementing the methods and/or recording results. Examples of devices that may be used include, but are not limited to, electronic computing devices, including computers of all types. When the methods described herein are implemented and/or recorded in a computer, any computer-readable medium capable of containing a computer program can contain the computer program that can be used to configure the computer to perform the steps of the methods. Examples of computer readable media that can be used include, but are not limited to, diskettes, CD-ROMs, DVDs, ROMs, RAMs, non-transitory computer readable media, and other memory and computer storage devices. Computer programs usable to configure a computer to perform the steps of the methods, assemble sequence information, and/or record results may also be provided over an electronic network, such as over the internet, an intranet, or other network.
The "consumable sequencing card" (FIG. 24) can be incorporated into fluorescence imaging equipment known in the art. Any fluorescence microscope with many varying characteristics can perform this sequencing read. For example: wide field lamps, lasers, LEDs, multi-photon, confocal or total internal reflection illumination may be used for excitation and/or detection. On the emission detection channel of the fluorescence microscope, it is possible to have a camera(s) and/or photomultiplier(s) based on filter or grating based spectral resolution (one or more spectrally resolved emission wavelengths). A standard computer can control the consumable sequencing card, the reagents flowing through the card and the detection by fluorescence microscopy.
Sequencing data can be analyzed by any number of standard next generation sequencing assemblers (see, e.g., Wajid and Serpidin, "Review of genetic algorithm resources for genetic algorithms for next generation sequences"Genomics, proteomics & bioinformatics,10 (2), 58-73, 2012). Sequencing data obtained within a single diffraction-limited region of the microscope is "locally assembled" to generate consensus sequences from multiple reads within the diffraction spot. The reads assembled from multiple diffraction points are then mapped together to produce a contiguous sequence representing the entire set of targeted genes, or de novo assembly of the whole genome.
Additional teachings related to the present invention are described in one or more of the following: u.s. 8,148,512, u.s. 7,473,767, u.s. 7,919,237, u.s. 7,941,279, u.s. 8,415,102, u.s. 8,492,094, u.s. 8,519,115, u.s. 2009/0220978, u.s. 2009/0299640, u.s. 2010/0015607, u.s. 2010/0261026, u.s. 2011/0086774, u.s. 2011/0145176, u.s. 2011/0201515, u.s. 2011/0229888, u.s. 2013/0004482, u.s. 2013/0017971, u.s. 2013/0178372, u.s. 2013/0230851, u.s. 2013/0337444, u.s. 2013/0345161, u.s. 2014/0005067, u.s. 2014/0017688, u.s. 2014/0037620, u.s. 2014/0087959, u.s. 2014/0154681, and u.s. 2014/0162251, each of which is incorporated herein by reference in its entirety.
Examples
Example 1: the method of the present invention for sequencing a target nucleic acid is rapid
The following describes the timing of the steps in the method of the present invention and as shown in figures 8 to 12.
The present invention requires minimal sample preparation. For example, as shown in fig. 13, the nucleic acids in the sample can begin to be read after 2 hours or less or a preparation time; this is significantly less time than required for Ion Torrent (AmpliSeq;) or illumina (trusight) sequencing, which requires about 12 or 9 hours of preparation time, respectively.
The calculation of an exemplary run is shown in fig. 14, and the calculation of the cycle time is shown in fig. 15.
Binding of the probe population to the immobilized target nucleic acid takes about 60 seconds. This reaction can be accelerated by using multiple copies of the target binding domain on the synthetic backbone. With microfluidically controlled fluid exchange devices, it takes about half a second to wash away unbound probes.
It takes about 15 seconds to add the first set of complementary nucleic acids (comprising detectable labels) and bind it to the attachment region in the first position of the barcode domain.
Each field of view (FOV) is imaged for four different colors, each color representing a single base. Fiducials placed on either the 5 'capture probe or the 3' capture probe (or both) can facilitate reading only those optical barcodes that are aligned between the two locations (consistent with the presence of a nicked target nucleic acid). Fiducials may also be added to each field of view to produce equal alignments of images after successive steps in the sequencing process. All four images can be acquired in a single FOV and then the optical reading device can be moved to the new FOV or take all FOVs in one color and then re-image in a second color. A single FOV can be read in about half a second. It takes about half a second to move to the next FOV. Therefore, the time to read "n" FOVs is equal to "n" x1 sec).
Complementary nucleic acids with detectable labels are removed from the first position of the barcode domain by heating or washing with an excess of complementary nucleic acids lacking a detectable label. If desired, the rate of exchange of the detectable label can be accelerated by incorporating small single-stranded oligonucleotides that accelerate the rate of exchange of the detectable label (e.g., "Toe-Hold" probes; see, e.g., Seeling et al), “Catalyzed Relaxation of a Metastable DNA Fuel”; J. Am. Chem. Soc2006, 128(37), pp 12211-. The FOV can be re-imaged to confirm that all complementary nucleic acids with detectable labels are removed before continuing to move. This takes about fifteen seconds. Can weighThis step is repeated until the background signal level is reached.
The above steps or the remaining positions in the barcode field of the probe are repeated.
The total time of reading is equal to m (base reading) × (15 seconds + n FOV × 1 second + 15 seconds). For example, when the number of positions in the barcode domain is 6 and 20 FOVs, the time of reading is equal to 6X (30 + 20 + 15) or 390 seconds.
The probes of the first population are de-hybridized. This takes about sixty seconds.
The above steps are repeated for the second and subsequent probe populations. If the population of sequencing probes is organized by melting temperature (Tm), multiple hybridizations will be required for each probe population to ensure that each base is covered to the required depth (this is driven by the error rate). Furthermore, by analyzing the hybridization reads during the run, each individual gene that is well sequenced can be identified before the entire sequence is actually determined. Thus, the cycle may be repeated until a particular desired error frequency (or coverage) is met.
Using the above timing sequences, together with some estimates of the binding density of gapped nucleic acids, the throughput of the Nanostring (NSTG) -next generation sequencer of the present invention can be estimated.
The net throughput of the sequencer is given by:
fractional base occupancy X < gap length > X number of gaps per FOV X number of bases per optical barcode/[ 60 seconds (probe hybridized to target nucleic acid) + 0.5 seconds (wash) + m: number of positions in barcode domain X (15 seconds (complementary nucleic acid bound) + nfovsX 1+ 15 seconds (complementary nucleic acid unbound)) + 60 seconds (probe unhybridization to target nucleic acid) ].
Thus, in one example, the total "cycles" of a single gapped nucleic acid (taken together from the method shown in figure 10):
60 seconds (probe to target nucleic acid hybridization) + 0.5 seconds (wash) + m-base X (15 seconds (bound complementary nucleic acid) + nFOV X1 + 15 seconds (unbound complementary nucleic acid)) + 60 seconds (probe to target nucleic acid hybridization). Using m = 6, nFOV = 20, generation time = 60 + 0.5 + 390 + 60 = 510.5 seconds.
Suppose that: the 1% occupancy of the nucleic acid region of the notch, 4000 bases per notch, and 5000 notches of the nucleic acid fragment per FOV, and m of 6 and nFOV of 20 (as described above) produced the following net fluxes:
0.01X 4000X 5000X 20 = 4,000,000 6-base reads per 510.5 seconds = 47,012.73 bases/second.
Thus, in this example, the net flux measured continuously every 24 hours = 4.062 gigabases (Gb) per day. The surrogate estimates are up to 12 Gb per day. See fig. 12.
As shown in fig. 14, the run time required to sequence 100 different target nucleic acids ("100-plex") was about 4.6 hours; the run time required to sequence 1000 different target nucleic acids ("1000-plex") was about 16 hours.
FIG. 16 compares the sequencing rates, reads, and clinical utility of the present invention with various other sequencing methods/devices.
Example 2: the method of the invention has a low error rate
Figure 17 shows that the present invention has a raw error rate of about 2.1% when the tip position is omitted.
For the claimed invention, the error rate associated with sequencing is related to the free energy difference between the perfectly matched (m + n) -mer and the single base mismatched (m-1+ n) -mer. The sum of m + n is the number of nucleotides in the target binding domain, and m represents the number of positions in the barcode domain. An equation can be used to estimate hybridization selectivity (see Owczarzy, R. (2005), Biophys. chem., 117:207-www) idtdna.com/analyzer/Applications/Instructions/Default.aspxAnalyzerDefinitions=true#MismatchMeltTemp)：
Wherein KaIs a binding equilibrium constant obtained from a predicted thermodynamic parameter,
theta represents the percentage of binding between the exact complementary sequence and the single base mismatch sequence that is expected to anneal to the target at the specified hybridization temperature. T is the hybridization temperature in Kelvins,h ° (enthalpy) ands ° (entropy) is the melting parameter calculated from the sequence and published nearest neighbor near thermodynamic parameters, and R is the ideal gas constant (1.987 cal. K.)-1mole-1) [ chain 1/2 ]]Is the molar concentration of the oligonucleotide, and a constant of-273.15 converts the temperature from Kelvin to degrees Celsius. DNA/DNA base pairs were obtained from the following publications (see, Allawi, h., santallucia, J.Biochemistry36, 10581), RNA/DNA base pairs (see, Sugimoto et al,Biochemistry34, 11211-6), RNA/RNA base pairs (see, Xia, t. et al,Biochemistry37, 14719).
An example of an estimate of the approximate error rate expected from an NSTG sequencer is as follows. For (m + n) equal to the 8' mer. Consider the following 8-mer barcodes and their single base mismatches.
(region of sequence)
(for perfect match optical bar code sequencing)
(sequencing optical barcodes with single base mismatch (G-T) pairing).
Using an IDT calculator based on the above equation yields:
at 17.4 ℃ (Tm for perfect match case), (50%/0.3%) would be the ratio of correct optical barcode to incorrect barcode that hybridized to the sequence at Tm, giving an estimated error rate for the sequence of 0.6%.
Very high GC content sequencing calculations yielded:
(region of sequence)
(for perfect match optical bar code sequencing)
(sequencing optical barcodes with single base mismatch (G-A) mismatches).
At 41.9 ℃ (Tm for perfect match case), (50%/0.4%) would be the ratio of correct optical barcode to incorrect barcode that hybridized to the sequence at Tm, giving an estimated error rate for the sequence of 0.8%.
Examination of the number of 8 mer pairs resulted in an error rate distribution in the range of 0.2% to 1%. Although the above calculations differ from the conditions used, these calculations provide an indication that the method of the invention will have a relatively low inherent error rate when compared to other single molecule sequencing techniques, such as the Pacific Biosciences and Oxford Nanopore technique, where the error rate can be significant (> 10%).
FIG. 18 shows that the original accuracy of the present invention is higher than other sequencing methods. Thus, the present invention provides a consensus sequence from a single target after fewer passes than are required for other sequencing methods. Furthermore, the present invention can achieve "perfect identity/no error" sequencing (i.e., 99.9999%/Q60) after 30 or more passes, whereas the PacBio sequencing method (for example) cannot achieve such identity after 70 passes.
Example 3: the invention has single base pair resolution capability
Figure 19 shows that the present invention has single base resolution and has a low error rate (in the range of 0% to 1.5% depending on the specific nucleotide substitution).
Additional experiments were performed using target RNA that was hybridized to barcodes and immobilized to the surface of the cassette using normal NanoString gene expression binding techniques (see, e.g., for,Geiss et al, "Direct multiplexed measurement of gene expression with color-coded probe pages";Nature Biotechnology26, 317-. Longer lengths of target binding domains give higher counts. It also shows that the 10-mer target binding domain is sufficient to register sequences above background. Each of the matches of individual single base changes was synthesized with a surrogate optical barcode. The ratio of correct to incorrect optical barcodes was counted (figures 24 and 25).
The ability of the 10-mer to detect SNPs, true sequences >15000 counts above background, while incorrect sequences > 400 counts above background at the most. In the presence of the correct probe, the expected error rate was 3% of the actual sequence. Note that this data is (essentially) a worse case. Only 10 base pair hybrid sequences were attached to a 6.6 kilobase optical barcode reporter (type Gen 2). No special condition optimization was performed. However, this data does indicate that NanoString next generation sequencing methods are able to resolve single base pairs of sequences.
The detailed materials and methods utilized in the above studies were as follows:
hybridization protocol Probe B plus code set
Get 25ul of the elements (194 code set)
Add 5ul Probe B + complement to target (100uM)
Addition of 15ul Hyb buffer (14.56 XSSPE 0.18% Tween 20)
SSPE (150mM NaCl, NaH2PO4xH2O 10mM, Na2EDTA 10mM)
Incubate on ice for 10 min
Add 150ul G beads (40ul 10mg/ml G beads plus 110 ul 5 XSSPE 0.1% Tween 20)
Incubate at room temperature for 10 min
Three washes with 0.1SSPE 0.1% Tween 20 using a magnetic collector
Elute in 100ul 0.1x SSPE for 10 minutes at 45 ℃.
Target hybridization protocol (750mM NaCl)
20ul of the above elution sample was taken
Addition of 10ul hyb buffer
Addition of 1ul target (100nM biotinylated RNA)
Incubate on ice for 30 min
15ul were taken and bound to a streptavidin slide for 20 minutes, the fluid was pulled with a G hook and counted with nCounter.
Material
Element 194 codeset
Oligonucleotides were purchased from IDT
SSPE (150mM NaCl, NaH2PO4xH2O 10mM, Na2EDTA 10mM)
Hyb buffer (14.56 XSSPE 0.18% Tween 20).
Table 2: 12. 11, 8-mer probe B sequence. (SEQ ID NO: 30-34)
Table 3: target sequence (in bold; SEQ ID NO: 35)
Table 4: 10-mer mismatched Probe B sequences (in bold; SEQ ID NO: 36 through SEQ ID NO: 41)
Sequence listing
<110> NanoString Technologies, Inc.
BEECHEM, Joseph
KHAFIZOV, Rustem
<120> sequencing without enzyme and amplification
<130> NATE-025/001
<150> US 62/082,883
<151> 2014-11-21
<160> 104
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 1
atacatctag 10
<210> 2
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 2
gatctacata 10
<210> 3
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 3
ttaggtaaag 10
<210> 4
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 4
tcttcattac 10
<210> 5
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 5
atgaatctac 10
<210> 6
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 6
tcaatgtatg 10
<210> 7
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 7
aattgagtac 10
<210> 8
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 8
atgttaatgg 10
<210> 9
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 9
aattaggatg 10
<210> 10
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 10
ataatggatc 10
<210> 11
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 11
taataaggtg 10
<210> 12
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 12
tagttagagc 10
<210> 13
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 13
atagagaagg 10
<210> 14
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 14
ttgatgatac 10
<210> 15
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 15
atagtgattc 10
<210> 16
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 16
tataacgatg 10
<210> 17
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 17
ttaagtttag 10
<210> 18
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 18
atacgttatg 10
<210> 19
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 19
tgtactatag 10
<210> 20
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 20
ttaacaagtg 10
<210> 21
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 21
aactatgtac 10
<210> 22
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 22
taactatgac 10
<210> 23
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 23
actaatgttc 10
<210> 24
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 24
tcattgaatg 10
<210> 25
<211> 14
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 25
ctgtctcatc tctt 14
<210> 26
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 26
ctgtctcatc tcttgctgca tcctgt 26
<210> 27
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 27
ctgtctcatc tcttgctgca tcctgtcggt tcacgttg 38
<210> 28
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 28
ctgtctcatc ttgctgcatc ctgtcggttc acgttg 36
<210> 29
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 29
ctgtctcatt ttgctgcatc ctgtccgttc acgttg 36
<210> 30
<211> 43
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 30
gactgtaccc acgcgatgac gttcgtcaag agtcgcataa tct 43
<210> 31
<211> 44
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 31
agactgtacc acaagaatcc ctgctagctg aaggagggtc aaac 44
<210> 32
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 32
gagactgtac cctacgtata tatccaagtg gttatgtccg acggc 45
<210> 33
<211> 46
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 33
tgagactgta ccacccctcc aaacgcattc ttattggcaa atggaa 46
<210> 34
<211> 47
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 34
ctgagactgt acccgggaat cggcatttcg cattcttagg atctaaa 47
<210> 35
<211> 44
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 35
caatgtgagt ctcttggtac agtctcagtt agtcactccc taag 44
<210> 36
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 36
gagacagtac cctggtctag gtatctaatt cgtgggtcgg gtact 45
<210> 37
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 37
gagaccgtac cgctcatttt gaacatacga ttgcgattac ggaaa 45
<210> 38
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 38
gagacggtac cttaaagcta tccacgaatg tcaaaaatgt ggttt 45
<210> 39
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 39
gagagtgtac ccaatgcttg cagtatgtat cctgatcgtg cgtgc 45
<210> 40
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 40
gagaatgtac cctcatacca atgtaaagta tagttaacgc cctgt 45
<210> 41
<211> 45
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 41
gagattgtac cctacatata taggaaaagg gaaggtagaa gagct 45
<210> 42
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 42
cacgaacgtc ag 12
<210> 43
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 43
catcgcatgc ct 12
<210> 44
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 44
gtcatctcct ac 12
<210> 45
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 45
gtcatccgct ac 12
<210> 46
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 46
gtcatcgact ac 12
<210> 47
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 47
gtcatcttct ac 12
<210> 48
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 48
gtcatcacct ac 12
<210> 49
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 49
gtcatcactc ac 12
<210> 50
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 50
gtcatcttcg ac 12
<210> 51
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 51
gtcatcaact ac 12
<210> 52
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 52
gtcatccgta ac 12
<210> 53
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 53
gtcatccgaa ac 12
<210> 54
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 54
gtcatcacaa ac 12
<210> 55
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 55
gtcatcttgc ac 12
<210> 56
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 56
gtcatcttgc ct 12
<210> 57
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 57
gtcatccgtc ct 12
<210> 58
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 58
cttttcacct ct 12
<210> 59
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 59
cttttcctct ct 12
<210> 60
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 60
cttttcgact ct 12
<210> 61
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 61
cttttctgct ct 12
<210> 62
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 62
cttttctgta ct 12
<210> 63
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 63
cttttctgtg ct 12
<210> 64
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 64
cttttctgtc ct 12
<210> 65
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 65
cttttcactc ct 12
<210> 66
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 66
cttttcgttc ct 12
<210> 67
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 67
cttttcgtac ct 12
<210> 68
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 68
cttttccgtc ct 12
<210> 69
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 69
cttttctgac ct 12
<210> 70
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 70
aggcatgcga tg 12
<210> 71
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 71
aggcattgtg ct 12
<210> 72
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 72
aggcattgct ct 12
<210> 73
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 73
aggcatttct ac 12
<210> 74
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 74
aggcatacct ac 12
<210> 75
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 75
aggcatttgc ac 12
<210> 76
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 76
aggcatcgtc ct 12
<210> 77
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 77
tcctgtcggt tc 12
<210> 78
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 78
gttcaatgct ct 12
<210> 79
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 79
attcggtgct ct 12
<210> 80
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 80
gatgcctgct ct 12
<210> 81
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 81
tttgcttgct ct 12
<210> 82
<211> 100
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 82
ttcactgtag ctgtctcatt ttgctgcatc ctgtccgttc acgttggagc ttgtcatccg 60
tcctcttttc actcctaggc atttgcctat tcggcgtcct 100
<210> 83
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 83
cgatctggtt 10
<210> 84
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 84
cgatctggtt 10
<210> 85
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 85
gctagaccaa 10
<210> 86
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 86
gctggaccaa 10
<210> 87
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 87
gctcgaccaa 10
<210> 88
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 88
gcttgaccaa 10
<210> 89
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 89
gagactgtac 10
<210> 90
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 90
gagacagtac 10
<210> 91
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 91
gagaccgtac 10
<210> 92
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 92
gagacggtac 10
<210> 93
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 93
gagagtgtac 10
<210> 94
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 94
gagaatgtac 10
<210> 95
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 95
gagattgtac 10
<210> 96
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 96
gagactgtac 10
<210> 97
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 97
gagattgtac 10
<210> 98
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 98
gagaccgtac 10
<210> 99
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 99
gagagtgtac 10
<210> 100
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 100
ctgagactgt ac 12
<210> 101
<211> 11
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 101
tgagactgta c 11
<210> 102
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 102
gagactgtac 10
<210> 103
<211> 12
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 103
catgtcagag tc 12
<210> 104
<211> 11
<212> DNA
<213> Artificial sequence
<220>
<223> synthetic polynucleotide
<400> 104
catgtcagag t 11
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