WO2014143157A1 - Compositions and methods for long insert, paired end libraries of nucleic acids in emulsion droplets - Google Patents

Compositions and methods for long insert, paired end libraries of nucleic acids in emulsion droplets Download PDF

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WO2014143157A1
WO2014143157A1 PCT/US2013/060990 US2013060990W WO2014143157A1 WO 2014143157 A1 WO2014143157 A1 WO 2014143157A1 US 2013060990 W US2013060990 W US 2013060990W WO 2014143157 A1 WO2014143157 A1 WO 2014143157A1
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5phos
nucleic acid
unique
detectable oligonucleotide
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PCT/US2013/060990
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French (fr)
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Scott Steelman
Robert Nicol
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The Board Institute, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Definitions

  • the invention is directed to methods for uniquely labeling populations of nucleic acids of interest in emulsion droplets using random combinations of oligonucleotides.
  • the invention provides methods and compositions for uniquely labeling nucleic acids, such as DNA, peptides or proteins.
  • nucleic acids such as DNA, peptides or proteins.
  • One of the major limitations of prior art labeling techniques is the limited number of available unique labels. Typically the number of nucleic acids to be labeled in any given application far exceeds the number of unique labels that are available.
  • the methods of the invention can be used to synthesize essentially an infinite number of unique labels. Moreover, because of their nature, the labels can be easily detected and distinguished from each other, making them suitable for many applications and uses.
  • the methods of the invention also provides for amplifying nucleic acids to increase the number of read pairs properly mated via their unique index combination. Additionally, the methods of the invention allow for each end-labeled nucleic acid to be identically labeled at its 5' and 3' ends.
  • a method for labeling a nucleic acid at both its 5 ' and 3' ends with a unique label comprising the steps of:
  • each detectable oligonucleotide tag is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of said nucleic acid,
  • each end-labeled nucleic acid is identically labeled at its 5' and 3' ends.
  • nucleic acids [0007] Also provided are labeled nucleic acids and libraries of said nucleic acids.
  • Figure 1 shows that the efficiency of DNA circularization (cyclization) decreases as fragment length increases.
  • Figure 2 is a schematic depicting the technique for polymerase mediated index addition.
  • Figure 3 is a gel electrophoresis showing ligation of an adapter sequence in either a tube (T) or an emulsion droplet (E).
  • Figure 4 is a schematic depicting the technique for ligation mediated index addition.
  • Figure 5 is a schematic depicting the technique for symmetric ligation-mediated index addition.
  • Figure 6 is a flowchart detailing the criteria for barcode sequence selection.
  • Figure 7 is a schematic depicting the methodology for informatically deriving mate pairs.
  • Figure 8 shows catalyzing ligation by the controlled addition of MgCl 2 .
  • Figure 9 shows a stability determination of MgCl 2 in droplets.
  • Figure 10 shows a determination of the optimal ratio of genomic Index:genomic DNA.
  • Figure 11 is a schematic depicting the process of symmetric indexing in emulsion.
  • Figure 12 shows a proof of concept experiment.
  • Figure 13 shows an analysis of E. coli proof of concept libraries.
  • Figure 14 shows an analysis of lambda proof of concept libraries.
  • Figure 15 shows a determination of symmetry of indexing in E. coli proof of concept libraries.
  • Figure 16 is a schematic of a mate pair synthesis process using single stranded genomic DNA as the agent.
  • Figure 17 is a schematic of a mate pair synthesis using droplets and Nextera transposomes as detectable tags.
  • Figure 18 shows the determination of uniformity of blunt-ended indexing.
  • Figure 19 shows impact of ligation efficiency on bioinformatics end association.
  • Figure 20 shows a redesign of index sequences.
  • Figure 21 shows uniformity of indexing.
  • Figure 22 shows amplification of fragment ends via transposome-based selection.
  • Figures 23a and 23b illustrate enrichment of ends via in vitro transcription.
  • Figure 24 shows amplification of ends via anchored PCR.
  • the methods of the invention easily and efficiently generate libraries of unique labels.
  • libraries may be of any size, and are preferably large libraries including hundreds of thousands to billions of unique labels.
  • the libraries of unique labels may be synthesized separately or may be synthesized in real-time (e.g., while in the presence of the nucleic acid). Methods for nucleic acid sequencing and detection of non-nucleic acid detectable moieties are known in the art and are described herein.
  • the methods of the invention label nucleic acids in emulsion droplets.
  • the nucleic acid is identically labeled at its 5' and 3' ends. Further, the nucleic acids are amplified by a method such as, for example, anchored PCR.
  • the invention further provides for methods for creating mate-pair libraries of uniquely labeled nucleic acids such as genomic DNA fragments.
  • mate-pair although specific to certain next generation sequencing technologies, is intended to generically describe a "jumping" library.
  • a jumping library is any DNA construct where the physical genomic distance between sequencing reads can be derived without the need to sequence the entire intervening length of DNA.
  • some terms frequently used to describe such libraries include (but are not limited to): jumping libraries, distance libraries, long range libraries, linking libraries, long distance linking libraries, mate-pair libraries and long paired-end libraries.
  • the invention contemplates that the labels are prepared by sequentially attaching randomly selected oligonucleotides (referred to interchangeably as oligonucleotide tags) to each other.
  • oligonucleotide tags referred to interchangeably as oligonucleotide tags
  • the order in which the oligonucleotides attach to each other is random and in this way the resultant label is unique from other labels so generated.
  • the invention is based, in part, on the appreciation by the inventors that a limited number of oligonucleotides can be used to generate a much larger number of unique labels. The invention therefore allows a large number of labels to be generated (and thus a large number of nucleic acids to be uniquely labeled) using a relatively small number of oligonucleotides.
  • the unique labeling strategies of the invention provide methods for generating mate pairs from genomic DNA fragments of virtually any length. This is a significant advantage over the mate pair methods of the prior art which require circularization of the genomic DNA fragment and thus are limited by the length of the fragment. In contrast, the methods of the invention do not rely on circularization of the genomic DNA fragments and thus are able to generate mate pairs from genomic DNA fragments of various lengths.
  • Figure 1 shows that the efficiency of DNA circularization (cyclization) decreases as fragment length increases.
  • B Portion of the graph presented in panel A focused on the lkb to 12kb size range.
  • Figure 2 shows the technique for polymerase mediated index addition.
  • genomic DNA is size selected, end repaired and A-tailed.
  • the biotinyalted adapter can only ligate in one orientation on each end of the genomic DNA due to the presence of the T-tail.
  • the adapter is a partial duplex to allow for primer annealing in the next step of the process.
  • Multiple index libraries are created in emulsion and a polymerase-driven fill-in reaction is used to add the index.
  • the index libraries may contain some or all of the key components of the fill in reaction (i.e., MgCl 2 , dNTP, polymerase).
  • the library is comprised of unique single stranded DNA oligonucleotides (oligos). Each oligo will contain 3 distinct moieties: sequence complimentary to adapter (Ad), a unique index sequence (Idx) and a sequence used to "capture” the next index oligo which contains one or more dUTP nucleotides (B'/C). DNA is diluted to a desired concentration to control the number of molecules per droplet and is merged with the index droplet library. (4) A fill-in reaction is performed creating the complement to the index and the "capture" site.
  • Figure 3 is a gel electrophoresis showing ligation of an adapter sequence in either a tube (T) or an emulsion droplet (E).
  • Figure 4 is a schematic of the technique for ligation mediated index addition.
  • Genomic DNA is size selected, end repaired and A-tailed.
  • the biotinyalted adapter can only ligate in one orientation on each end of the genomic DNA due to the presence of the T-tail.
  • the adapter may be blunt ended or it may be a partial duplex with a sequence specific cohesive overhang to allow for index annealing in the next step of the process.
  • Multiple index libraries are created in emulsion and a ligation reaction is performed to add the index.
  • the index libraries may contain some or all of the key components of the ligation reaction (i.e., MgCl 2 , dNTP, ligase).
  • Each droplet will contain many copies of an individual (unique) index sequence.
  • DNA is diluted to a desired concentration to control the number of genomic fragments per droplet and is then joined to the index library allowing the ligation reaction to occur. Following ligation, the emulsion is broken and DNA is pooled, purified and prepared so that it can accept the next index. (4-6) The process of DNA dilution, index addition, ligation, pooling, clean-up and phosphorylation is repeated for the desired number of cycles, each time adding one new index to the end of the DNA. (7) After the final index addition, DNA is fragmented and the ends are collected via streptavidin beads. All fragments are ligated to technology specific sequencing adapters and ends are informatically paired based on their unique string of indexes.
  • Figure 5 depicts the technique for symmetric ligation-mediated index addition.
  • Genomic DNA is size selected, end repaired and A-tailed.
  • the biotinyalted adapter can only ligate in one orientation on each end of the genomic DNA due to the presence of the T-tail.
  • the adapter may be blunt ended or it may be a partial duplex with a sequence specific cohesive overhang to allow for index annealing in the next step of the process.
  • Multiple index libraries are created and a ligation reaction is performed to add the index. Following ligation, DNA molecules are pooled, purified and prepared for the next round of ligation. (4) This process is repeated Y number of times.
  • the total "diversity" of the population of unique index combinations is dictated by the number of indexes used raised to the power of the number of cycles performed. For example, 3 round of index ligation using a 1152 element array creates 1152 or 1,528,823,808 combinations. (5) After the final index addition, DNA is fragmented and the ends are collected via streptavidin beads. (6) Sheared DNA fragments are end repaired as needed and ligated to technology specific sequencing adapters. Following sequencing, the ends are informatically paired based on their unique string of indexes.
  • Figure 6 is a flowchart detailing the criteria for barcode sequence selection.
  • Figure 7 provides the methodology for informatically deriving mate pairs.
  • Left Panel For a standard Ilumina mate-pair library, each construct is sequenced using two reads. Since both ends of the parent genomic DNA fragment are contained in a single construct, two reads (i.e., a single read pair) are sufficient to establish a mate-pair.
  • Right Panel Unlike a standard library, a symmetrically indexed library requires a total of 4 reads (i.e., 2 read pairs) in order to establish a mate-pair. For a given construct, one read will contain the index information while the other is genomic DNA (i.e, one read pair defines one "half of the mate-pair). For each construct in the library, the algorithm must search the data set to identify appropriately matching indexing combinations. Once the index combinations are matched, their corresponding genomic reads can be positioned relative to the genome.
  • Figure 8 shows catalyzing ligation by the controlled addition of MgCl 2 .
  • MgCl 2 In order to prevent spontaneous ligation (concatamerization) of genomic DNA fragments, it is necessary to prepare the DNA in a solution that lacks one or more of the key components necessary to catalyze a ligation reaction. Due to stability and cost issues, sequestration of MgCl 2 in the index droplets would be preferable over other key components (i.e., ligase enzyme and ATP).
  • a single 380bp restriction fragment was generated from pBR322 plasmid DNA and prepared it such that it can only ligate to itself in one orientation. Thus, a 760bp ligated product is easily distinguishable from the unligated 380bp product.
  • a 10X modified ligation buffer lacking MgCl 2 was prepared but containing 500mM Tris-HCl pH 7.5, lOOmM dithiothreitol and lOmM ATP.
  • lanes 1-3 and lanes 7 and 8 reactions were prepared as indicated, incubated for 1 hour at room temperature, purified and run on an Agilent DNA 1000 chip.
  • Figure 9 shows stability determination of MgCl 2 in droplets.
  • Droplets containing 50mM, lOmM or ImM concentrations of MgCl 2 were prepared and stored at +4°C for ⁇ 4.5 days. Droplets were then broken and the aqueous phase was collected from each droplet "library” and transferred into fresh 1.5ml tubes.
  • Ligation reactions containing IX Modified Ligation buffer (i.e., buffer lacking MgCl 2 ), T4 DNA ligase and 380bp control fragment DNA were prepared.
  • Aqueous phase recovered from the various droplet libraries (lanes 1-3) or non-emulsified MgCl 2 (lanes 4-6) was added to the various ligation reactions.
  • the 50mM and lOmM reactions appeared to perform equally well while a marked reduction in the amount of ligated product was observed for the ImM condition.
  • the MgCl 2 released from the droplet library (lanes 1 -3) appeared equally capable of catalyzing the ligation reaction as freshly added MgCl 2 (lanes 4-6).
  • the 50mM droplet condition looks slightly less intense on the gel image due to slightly less material being loaded on the gel for that lane.
  • Figure 10 represents a determination of the optimal ratio of genomic Index: genomic DNA.
  • Lambda genomic DNA was sheared to a mean size of ⁇ 300bp using a Covaris S2 instrument. The genomic DNA was then end repaired and utilized in a ligation reaction containing variable molar ratios of Index:gDNA. Following index ligation, samples were end repaired, A-tailed and ligated to Illumina adapters. Samples were pooled and sequenced on an Illumina MiSeq. The percentage of reads where indexed was observed is shown. NOTE: The indexes used in this experiment were blunt ended 20bp sequences.
  • Figure 11 provides the process of symmetric indexing in emulsion.
  • Index libraries are prepared in an emulsion.
  • the droplets carrying index also contain a concentration of MgCl 2 such that when they are joined with a solution of DNA, ligase buffer and ligase enzyme, the final concentration of MgCl 2 in a given reaction is 50mM.
  • ligase buffer and ligase enzyme the final concentration of MgCl 2 in a given reaction is 50mM.
  • Figure 12 shows a proof of concept experiment. E.
  • coli genomic DNA was sheared to a mean size of approximately 300bp using a Covaris S2 instrument, end repaired, A-tailed and ligated to the cap adapter.
  • Lambda genomic DNA was prepared similarly, but was not sheared. Genomic DNA fragments were then subjected to 1, 2 or 3 rounds of blunt-ended index ligation in bulk (i.e., in microcentrifuge tubes). E. coli fragments were not sheared following index ligation while lambda fragments were sheared to approximately 500bp using a Covaris S2 instrument. Cap containing fragments were selected via incubation with paramagnetic streptavidin M-280 beads, end repaired, A-tailed and ligated to Illumina sequencing adapters. Samples were pooled and sequenced on an Illumina MiSeq using standard paired end chemistry.
  • FIG. 13 is an analysis of E. coli proof of concept libraries.
  • E. coli genomic DNA libraries were prepared in duplicate (Condi and Cond2) as described above (see Figure 12). Libraries were pooled and sequenced with a lOlbp paired read on a single MiSeq run. Paired reads that passed filter were analyzed together as a single population.
  • B The number of reads containing index at a given position are shown.
  • C The percent of reads containing index at a given position is shown.
  • Figure 14 provides an analysis of lambda proof of concept libraries.
  • Lambda phage genomic DNA libraries were prepared in duplicate (Condi and Cond2) as described above (see Figure 12). Libraries were pooled and sequenced with a lOlbp paired read on a single MiSeq run. Paired reads that passed filter were analyzed together as a single population.
  • C The percent of reads containing index at a given position is shown. The percentages shown are corrected for the fact that one half of the reads will necessarily be the genomic "end" of the library insert.
  • FIG 15 shows a determination of symmetry of indexing in E. coli proof of concept libraries.
  • E. coli genomic DNA was prepared as described above (see Figure 12). Libraries subjected to 1 (panels 1A and IB), 2 (panels 2A and 2B) or 3 (panels 3A and 3B) rounds of index ligation are shown. All data that passed filter was analyzed as read pairs which were then broken down into 20bp units (i.e., positions) and checked for the presence of index sequences. Positions where index sequences were detected are depicted by green boxes; positions where indexes were not detected are depicted by white boxes. The expected outcome for each library is denoted by a green asterisk.
  • Figure 16 is a schematic representation of a mate pair synthesis process using single stranded genomic DNA as the agent. Each droplet comprises both strands of the genomic fragment. As shown in the Figure, the strands are identically labeled at one end.
  • Figure 17 is a schematic of a mate pair synthesis using droplets and Nextera transposomes as detectable tags.
  • Figure 18 shows the determination of uniformity of blunt-ended indexing.
  • C57BL/6J mouse genomic DNA was sheared to approximately 40kb using a Genemachines Hydroshear. Samples were run on a 0.7% agarose gel and fragments of approximately 31kb and 38kb were collected and purified separately. All fragments were then end repaired, A-tailed and ligated to the biotinylated cap adapter. Fragments were then ligated to blunt-ended index sequences contained in a droplet library using the Raindance Thunderstorm instrument. Following each round of index ligation, the emulsion was broken and samples were end repaired and purified for use in subsequent rounds of ligation. A total of 3 rounds of index ligation were performed.
  • samples were sheared to ⁇ 500bp in length using a Covaris S2 instrument. Fragments containing the biotinylated cap adapter were selected using streptavidin M-280 beads, end repaired, A-tailed and ligated to Illumina sequencing adapters. Samples were then sequenced using an Illumina MiSeq instrument. The location of the cap sequence within a given read was determined. The total number of reads with cap sequence identified at a given position within the read are shown.
  • Figure 19 describes impact of ligation efficiency on bioinformatics end association.
  • Upon analysis of the indexed mouse libraries (see Figure 18), it was observed that clear populations of reads carrying 1, 2 or 3 indexes were present in the final library. The fact that multiple distinct populations were observed indicates a lack of symmetry in the indexing protocol. This presents a challenge to the informatic association of mate pairs since, for example, a read carrying 3 indexes may have its mate in the pool of reads carrying 3, 2 or 1 indexes. Thus, the likelihood of correctly pairing reads decreases as the number of indexes present decreases.
  • Figure 20 is a redesign of index sequences to improve ligation efficiency.
  • the cap adapter and all barcodes were redesigned to carry a 4-bp cohesive overhang on either side of the barcode (but only on one side of the cap adapter). Barcodes were separated into 4 different populations (A, B, C and D) depending on the sequence of the 4-bp cohesive overhang. The sequence of the cohesive overhang for each population is shown.
  • Figure 21 shows uniformity of indexing using cohesive overhang indexing.
  • E. coli and lambda genomic DNA libraries were prepared as described above (see Figure 12), but this time using cohesive overhang indexes in conjunction with a cohesive overhang ended cap adapter.
  • Reads were analyzed as before and the location of the biotinylated cap within the read was determined. The total number of reads with cap sequence identified at a given position within the read are shown. This analysis revealed a clear improvement in the homogeneity of the indexed read population where the vast majority of the reads from the cohesive overhang indexed libraries carried 3 indexes
  • Figures 22 to 24 show examples of fragment amplification techniques.
  • Figure 22 shows transposome-based selection and amplification of ends creating many fragments where both ends are flanked by an Illumina P5 sequence.
  • Figure 23 shows enrichment of ends via in vitro transcription wherein T7 RNA polymerase is used in order to amplify both ends of a given molecule.
  • Figure 24 shows amplification using an anchored PCR technique described in Example 3.
  • the invention provides a method comprising generating a plurality of unique labels by attaching at least two, randomly selected, detectable oligonucleotide tags to each other in a sequential manner, and associating each unique label with a separate nucleic acid.
  • the at least two detectable tags are attached to each other using ligation, polymerization, or a combination thereof.
  • the unique label is generated in an emulsion droplet or in a series of emulsion droplets.
  • the library of uniquely-labeled nucleic acids is generated using an emulsion droplet or a series of emulsion droplets.
  • the invention provides a method comprising sequentially attaching at least two detectable oligonucleotide tags to a 5' and/or 3' end of a first nucleic acid, wherein each detectable oligonucleotide tag is randomly selected from a plurality of detectable oligonucleotide tags, thereby generating a second nucleic acid comprising the first nucleic acid attached at its 5' and/or 3' end with a unique combination of detectable oligonucleotide tags.
  • the first nucleic acid is a genomic DNA fragment.
  • each end-labeled nucleic acid is (a) identically labeled at its 5' and 3' ends, and (b) uniquely labeled relative to other nucleic acids in the plurality, wherein each detectable oligonucleotide tags is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of a nucleic acid.
  • the number of oligonucleotides is 10-fold, 100-fold, 1000- fold, or 10000-fold less than the number of nucleic acids.
  • the method further comprises fragmenting end-labeled nucleic acids into at least a 5' fragment comprising the 5' end of the nucleic acid attached to the random combination of n oligonucleotide tags and into a 3' fragment comprising the 3' end of the nucleic acid attached to the random combination of n oligonucleotide tags.
  • the 5' and 3' fragments are about 10-1000 bases (base pairs) in length, or about 10-500 bases in length, or about 10-200 bases in length.
  • the method further comprises sequencing the 5' and 3' fragments.
  • the invention provides a method comprising (a) end-labeling two or more first subsets of nucleic acids with a detectable oligonucleotide tag to produce nucleic acids within a subset that are identically end-labeled relative to each other and uniquely end-labeled relative to nucleic acids in other subsets; (b) combining two or more subsets of uniquely end- labeled nucleic acids to form a pool of nucleic acids, wherein the pool comprises two or more second subsets of nucleic acids that are distinct from the two or more first subsets of nucleic acids; (c) identically end-labeling two or more second subsets of nucleic acids with a second detectable oligonucleotide tag to produce nucleic acids within a second subset that are uniquely labeled relative to nucleic acids in the same or different second subsets; and (d) repeating steps (b) and (c) until a number of unique end-l
  • the invention provides a method comprising (a) providing a pool of nucleic acids; (b) separating the pool of nucleic acids into sub-pools of nucleic acids; (c) end- labeling nucleic acids in each sub-pool of with one of mj detectable oligonucleotide tags thereby producing sub-pools of labeled nucleic acids, wherein nucleic acids in a sub-pool are identically end-labeled to each other; (d) combining sub-pools of labeled nucleic acids to create a pool of labeled nucleic acids; (e) separating the pool of labeled nucleic acid molecules into second sub- pools of labeled nucleic acids; (f) repeating steps (c) to (e) n times to produce nucleic acids end- labeled with n detectable oligonucleotide tags wherein the pool in (a) consists of a number of nucleic acids that is less than (mi)
  • the invention provides a method comprising (a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acid; (b) fusing each individual library droplet with a single index droplet from a plurality of mi index droplets, each index droplet comprising a plurality of one unique detectable oligonucleotide tag; (c) end-labeling the nucleic acid with the unique detectable oligonucleotide tag in a fused droplet; (d) harvesting end-labeled nucleic acids from the fused droplets and generating another population of library droplets comprising end-labeled nucleic acids; and (e) repeating steps (b) to (d) n times to produce nucleic acids end-labeled with n unique detectable oligonucleotide tag, wherein the n unique detectable oligonucleotide tags generate an (mi)(m 2 )(m 3 ).
  • end-labeling comprises ligation of the unique oligonucleotide tag with the nucleic acid.
  • the unique oligonucleotide tag is double- stranded.
  • the method further comprises phosphorylating the nucleic acids between steps (b) and (c).
  • end-labeling comprises a polymerase-mediated fill-in reaction.
  • the polymerase-mediated fill-in reaction comprises (a) producing a single-stranded cohesive overhang on the nucleic acid, wherein the cohesive overhang is complementary to one end of the unique detectable oligonucleotide tag; (b) annealing the complementary end of the unique oligonucleotide tag to the single-stranded cohesive overhang such that at least one nucleotide of the unique detectable oligonucleotide tag is not annealed to the nucleic acid, producing a unique detectable oligonucleotide tag cohesive overhang; and (c) extending the single-stranded cohesive overhang of (a) using a polymerase and nucleotides complementary to the unique detectable oligonucleotide tag cohesive overhang to produce a double-stranded unique detectable oligonucleotide tag.
  • the single-stranded cohesive overhang on the nucleic acid is produced by a USER enzyme.
  • the unique detectable oligonucleotide tag is single-stranded.
  • an oligonucleotide adapter is added to the nucleic acids before labeling with the unique detectable oligonucleotide tags.
  • the adapter comprises biotin.
  • the adapter comprises a thymidine tail cohesive overhang.
  • labeling occurs at the 5' and 3' ends of the nucleic acid. In some embodiments, labeling occurs at the 5' or the 3' end of the nucleic acid.
  • the nucleic acids are genomic DNA, cDNA, PCR products, or fragments thereof.
  • the method further comprises fragmenting uniquely end- labeled nucleic acids. In some embodiments, the method further comprises sequencing the uniquely end-labeled nucleic acids.
  • the number of nucleic acids in the pool is at least two times greater than the number of unique oligonucleotide tags.
  • the invention provides a method comprising (a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acids end-labeled on its 5' and 3' ends with oligonucleotide label, wherein the oligonucleotide label on the 5' end (the 5' oligonucleotide label) and the oligonucleotide on the 3' end (the 3' oligonucleotide label) comprise a nucleotide cohesive overhang, and wherein the nucleotide cohesive overhang on the 5' oligonucleotide label is complementary to the nucleotide cohesive overhang on the 3' oligonucleotide label; (b) fusing each individual library droplet with a droplet comprising a DNA fragmenting enzyme, thereby producing a fused droplet; (c) fragmenting the nucleic acid with the 5' and 3' oligonucleotide labels in
  • the 5' oligonucleotide label and/or the 3' oligonucleotide comprises a biotin label.
  • the method further comprises (e) sequencing the ligated nucleic acid.
  • the DNA fragmenting agent is Nextera.
  • the invention provides a method comprising (a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acid comprising an oligonucleotide adapter; (b) melting the nucleic acid; (c) fusing each individual library droplet comprising a melted nucleic acid with a single index droplet from a plurality of ml index droplets, each index droplet comprising a first unique single-stranded detectable oligonucleotide tag, wherein the first unique single-stranded detectable oligonucleotide tag comprises a region complementary to the oligonucleotide adapter; (d) annealing the first unique single-stranded detectable oligonucleotide tag to the nucleic acid and performing a fill-in reaction, thereby producing an end-labeled nucleic acid; (e) harvesting end- labeled nucleic acids from the fused droplets and generating
  • the invention provides a method comprising sequencing a pair of genomic nucleic acid fragments, wherein the genomic nucleic acid fragments are attached to identical unique labels at one of their ends that indicates the genomic nucleic acid fragments were separated by a known distance in a genome prior to fragmentation.
  • the pair of nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation.
  • the method further comprises generating the pair of genomic nucleic acid fragments by fragmenting nucleic acids comprising genomic sequence and identical non-genomic sequence at their 5' and 3' ends.
  • the invention provides a composition comprising a plurality of paired nucleic acid fragments attached to unique labels at one end, wherein paired nucleic acid fragments:
  • paired nucleic acid fragments were separated by greater than
  • paired nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation.
  • the invention provides a composition comprising a plurality of paired genomic nucleic acid fragments produced any of the foregoing methods.
  • the present invention further encompasses methods of making and/or using one or more of the embodiments described herein.
  • nucleic acid agent refers to a nucleic acid.
  • the nucleic acid agent may be single-stranded (ss) or double-stranded (ds), or it may be partially single-stranded and partially double-stranded.
  • Nucleic acid agents include but are not limited to DNA such as genomic DNA fragments, PCR and other amplification products, RNA, cDNA, and the like. Nucleic acid agents may be fragments of larger nucleic acids such as but not limited to genomic DNA fragments.
  • An agent of interest may be associated with a unique label.
  • "associated” refers to a relationship between the agent and the unique label such that the unique label may be used to identify the agent, identify the source or origin of the agent, identify one or more conditions to which the agent has been exposed, etc.
  • a label that is associated with an agent may be, for example, physical attached to the agent, either directly or indirectly, or it may be in the same defined, typically physically separate, volume as the agent.
  • a defined volume may be an emulsion droplet, a well (of for example a multiwell plate), a tube, a container, and the like. It is to be understood that the defined volume will typically contain only one agent and the label with which it is associated, although a volume containing multiple agents with multiple copies of the label is also contemplated depending on the application.
  • An agent may be associated with a single copy of a unique label or it may be associated with multiple copies of the same unique label including for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10,000, 100,000 or more copies of the same unique label.
  • the label is considered unique because it is different from labels associated with other, different agents.
  • Attachment of labels to agents may be direct or indirect.
  • the attachment chemistry will depend on the nature of the agent and/or any derivatisation or functionalisation applied to the agent.
  • labels can be directly attached through covalent attachment.
  • the label may include a moiety, which may be a non-nucleotide chemical modification, to facilitate attachment.
  • the label may include methylated nucleotides, uracil bases, phosphorothioate groups, ribonucleotides, diol linkages, disulphide linkages, etc., to enable covalent attachment to an agent.
  • a label can be attached to an agent via a linker or in another indirect manner.
  • linkers include, but are not limited to, carbon-containing chains, polyethylene glycol (PEG), nucleic acids, monosaccharide units, and peptides.
  • PEG polyethylene glycol
  • the linkers may be cleavable under certain conditions. Cleavable linkers are discussed in greater detail herein.
  • nucleic acids for attaching nucleic acids to each other, as for example attaching nucleic acid labels to nucleic acid agents, are known in the art. Such methods include but are not limited to ligation and polymerase-mediated attachment methods (see, e.g., U.S. Patent Nos. 7863058 and 7754429; Green and Sambrook. Molecular Cloning: A Laboratory Manual, Fourth Edition, 2012; Current Protocols in Molecular Biology, and Current Protocols in Nucleic Acid Chemistry, all of which are incorporated herein by reference).
  • the unique labels of the invention are, at least in part, nucleic acid in nature, and are generated by sequentially attaching two or more detectable oligonucleotide tags to each other.
  • a detectable oligonucleotide tag is an oligonucleotide that can be detected by sequencing of its nucleotide sequence and/or by detecting non-nucleic acid detectable moieties it may be attached to.
  • the oligonucleotides tags are typically randomly selected from a diverse plurality of oligonucleotide tags.
  • an oligonucleotide tag may be present once in a plurality or it may be present multiple times in a plurality.
  • the plurality of tags may be comprised of a number of subsets each comprising a plurality of identical tags.
  • these subsets are physically separate from each other. Physical separation may be achieved by providing the subsets in separate droplets from an emulsion. It is the random selection and thus combination of oligonucleotide tags that results in a unique label.
  • oligonucleotide refers to a nucleic acid such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA/RNA hybrids and includes analogs of either DNA or RNA made from nucleotide analogs known in the art (see, e.g. U.S.
  • Oligonucleotides may be single-stranded (such as sense or antisense oligonucleotides), double-stranded, or partially single-stranded and partially double-stranded.
  • a unique nucleotide sequence may be a nucleotide sequence that is different (and thus distinguishable) from the sequence of each detectable oligonucleotide tag in a plurality of detectable oligonucleotide tags.
  • a unique nucleotide sequence may also be a nucleotide sequence that is different (and thus distinguishable) from the sequence of each detectable oligonucleotide tag in a first plurality of detectable oligonucleotide tags but identical to the sequence of at least one detectable oligonucleotide tag in a second plurality of detectable oligonucleotide tags.
  • a unique sequence may differ from other sequences by multiple bases (or base pairs). The multiple bases may be contiguous or non-contiguous. Methods for obtaining nucleotide sequences (e.g., sequencing methods) are described herein and/or are known in the art.
  • detectable oligonucleotide tags comprise one or more of a ligation sequence, a priming sequence, a capture sequence, and a unique sequence (optionally referred to herein as an index sequence).
  • a ligation sequence is a sequence complementary to a second nucleotide sequence which allows for ligation of the detectable oligonucleotide tag to another entity comprising the second nucleotide sequence, e.g., another detectable oligonucleotide tag or an oligonucleotide adapter.
  • a priming sequence is a sequence complementary to a primer, e.g., an oligonucleotide primer used for an amplification reaction such as but not limited to PCR.
  • a capture sequence is a sequence capable of being bound by a capture entity.
  • a capture entity may be an oligonucleotide comprising a nucleotide sequence complementary to a capture sequence, e.g. a second detectable oligonucleotide tag.
  • a capture entity may also be any other entity capable of binding to the capture sequence, e.g. an antibody or peptide.
  • An index sequence is a sequence comprising a unique nucleotide sequence and/or a detectable moiety as described above.
  • “Complementary” is a term which is used to indicate a sufficient degree of complementarity between two nucleotide sequences such that stable and specific binding occurs between one and preferably more bases (or nucleotides, as the terms are used interchangeably herein) of the two sequences. For example, if a nucleotide in a first nucleotide sequence is capable of hydrogen bonding with a nucleotide in second nucleotide sequence, then the bases are considered to be complementary to each other. Complete (i.e., 100%) complementarity between a first nucleotide sequence and a second nucleotide is preferable, but not required for ligation, priming, or capture sequences.
  • Table 1 below provides examples of certain oligonucleotide tags of the invention:
  • Each unique label comprises two or more detectable oligonucleotide tags.
  • the two or more tags may be three or more tags, four or more tags, or five or more tags.
  • a unique label comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more detectable tags.
  • the tags are typically bound to each other, typically in a directional manner.
  • Ligation reactions include blunt end ligation and cohesive overhang ligation. In some instances, ligation may comprise both blunt end and cohesive overhang ligation.
  • a cohesive overhang is a single stranded end sequence (attached to a double stranded sequence) capable of binding to another single stranded sequence thereby forming a double stranded sequence.
  • a cohesive overhang may be generated by a polymerase, a restriction endonuclease, a combination of a polymerase and a restriction endonuclease, or a Uracil-Specific Excision Reagent (USERTM) enzyme (New England BioLabs Inc., Ipswich, MA) or a combination of a Uracil DNA glycosylase enzyme and a DNA glycosylase-lyase Exonuclease VIII enzyme.
  • a cohesive overhang may be a thymidine tail.
  • Polymerization reactions include enzyme-mediated polymerization such as a polymerase-mediated fill-reaction.
  • detection comprises determining the presence, number, and/or order of detectable tags that comprise a unique label.
  • Methods of sequencing oligonucleotides and nucleic acids are well known in the art (see, e.g., W093/23564, WO98/28440 and W098/13523; U.S. Pat. Nos. 5,525,464; 5,202,231; 5,695,940; 4,971,903; 5,902,723; 5,795,782; 5,547,839 and 5,403,708; Sanger et al., Proc. Natl. Acad. Sci.
  • the invention provides methods for generating unique labels.
  • the methods typically use a plurality of detectable tags to generate unique labels.
  • a unique label is produced by sequentially attaching two or more detectable oligonucleotide tags to each other.
  • the detectable tags may be present or provided in a plurality of detectable tags.
  • the same or a different plurality of tags may be used as the source of each detectable tag comprised in a unique label.
  • a plurality of tags may be subdivided into subsets and single subsets may be used as the source for each tag. This is exemplified in at least FIG. 1.
  • a plurality of tags may comprise 2, 3 , 4, 5, 6, 7, 8, 9, 10, 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 , or more tags.
  • the tags within a plurality are unique relative to each other.
  • the methods of the invention allow an end user to generate a unique label for a plurality of agents using a number of tags that are less (and in some instances far less) than the number of agents to be labeled.
  • the number of tags may be up to or about 10-fold, 10 -fold, 10 - fold, or 10 4 -fold less than the number of agents.
  • the number of agents to be labeled will depend on the particular application.
  • the invention contemplates uniquely labeling at least 10 3 , 10 4 , 10 5 , 10°, 10', 10°, 10% or 10 ⁇ or more agents.
  • the agent comprises a plurality of nucleic acids.
  • the plurality of nucleic acids comprises at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 s , 10 9 , or 10 10 nucleic acids.
  • agents, detectable tags, and resultant unique labels are all present in a contained volume and are thus physically separate from other agents, detectable tags, and resultant unique labels.
  • the contained volume is on the order of picoliters, nanoliters, or microliters.
  • the contained volume may be a droplet such as an emulsion droplet.
  • an agent is attached to the unique label (or label intermediate) directly or indirectly.
  • the droplets are ruptured (or broken) and their contents are pooled (and effectively mixed together).
  • the contents of the pool may be introduced, at limiting dilution, into another plurality of emulsion droplets each of which comprises a single detectable oligonucleotide tag (and optionally multiple copies of the oligonucleotide tag).
  • the droplets are again ruptured, and the process is repeated until a sufficient number of unique labels is generated.
  • a subset of the plurality of agents is present in the same container during attachment of a detectable label.
  • the plurality of agents is separated such that each agent in the plurality is in a separate container, e.g., an emulsion droplet.
  • the process of pooling and subsequently separating the plurality of agents is performed n number of times, wherein n is the number of times required to generate (mi)(m 2 )(m 3 )...(m n ) number of combinations of detectable oligonucleotide tags, wherein (m 1 )(m 2 )(m 3 )...(m n ) number of combinations of detectable oligonucleotide tags is greater than the number of the plurality of agents.
  • the invention provides a method comprising
  • each unique label comprises at least two detectable oligonucleotide tags.
  • the invention provides another method comprising
  • the invention provides another method comprising
  • each droplet comprises an agent
  • each detectable oligonucleotide tag droplet comprising a plurality of identical detectable oligonucleotide tag
  • the invention provides another method comprising
  • each droplet comprises more than one agent
  • each detectable oligonucleotide tag droplet comprising a plurality of identical detectable oligonucleotide tag
  • each droplet comprises more than one agent
  • Fluorescence-activated droplet sorting (FADS): Efficient microfluidic cell sorting based on enzymatic activity. Lab Chip, 9, 1850. 2009; M.M. Kiss, L. Ortoleva-Donnelly, N.R. Beer, J. Warner, C.G. Bailey, B.W. Colston, J.M. Rothberg, D.R. Link, and J.H. Leamon. High-throughput quantitative polymerase chain reaction in pico liter droplets. Anal Chem. 2008 December 1 ; 80(23): 8975-8981 ; Edd et al. Controlled encapsulation of single-cells into monodisperse picolitre drops.
  • a “droplet” or “emulsion droplet”, as used herein, is an isolated portion of a first fluid that is completely surrounded by a second fluid.
  • the first and second fluids are immiscible with each other.
  • the discontinuous phase can be an aqueous solution and the continuous phase can a hydrophobic fluid such as an oil or a fluorocarbon oil. This is termed a water in oil emulsion.
  • the emulsion may be an oil in water emulsion.
  • the first liquid, which is dispersed in globules is referred to as the discontinuous phase
  • the second liquid is referred to as the continuous phase or the dispersion medium.
  • the continuous phase can be an aqueous solution and the discontinuous phase is a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, or hexadecane).
  • a hydrophobic fluid such as an oil (e.g., decane, tetradecane, or hexadecane).
  • the droplets or globules of oil in an oil in water emulsion are also referred to herein as "micelles”, whereas globules of water in a water in oil emulsion may be referred to as "reverse micelles".
  • the droplets may be spherical or substantially spherical; however, in other cases, the droplets may be non-spherical.
  • droplet library or “droplet libraries” are also referred to herein as an “emulsion library” or “emulsion libraries.”
  • examples of droplet libraries are collections of droplets that have different contents, ranging from DNA, primers, etc.
  • the droplets range in size from roughly 0.5 micron to 500 micron in diameter, which corresponds to about 1 pico liter to 1 nano liter. However, droplets can be as small as 5 microns and as large as 500 microns.
  • the droplets are at less than 100 microns, about 1 micron to about 100 microns in diameter. The most preferred size is about 20 to 40 microns in diameter (10 to 100 picoliters).
  • the preferred properties examined of droplet libraries include osmotic pressure balance, uniform size, and size ranges.
  • Droplets can be generated by infusing aqueous samples comprising library elements, e.g., agents, detectable tags, or combinations thereof, at a perpendicular angle to opposing oil streams. Droplets can be contained within a microfluidic channel. Microfluidic channels and method for manufacturing microfluidic channels are known in the art (see, e.g., McDonald JC, et al. (2000) Fabrication of microfluidic systems in poly(dimethylsiloxane) Electrophoresis 21:27-40; Siegel AC, et al.
  • Droplets can be optionally merged. Merging can be accomplished, e.g., by passing an electrical field through a microfluidic channel to merge charged droplets, or by addition of a chemical that breaks emulsions.
  • a chemical that breaks emulsions See K. Ahn, J. Agresti, H. Chong, M. Marquez and D. A. Weitz, Appl. Phys. Lett., 2006, 88, 264105 and D Link, E Grasland-Mongrain, A Duri, F Sarrazin, Z Cheng, G Cristobal, M Marquez, and DA Weitz. Angew. Chem. Int. Ed. 2006, 45, 2556 -2560 as examples.)
  • Generation of unique labels may occur in part or entirely in emulsion droplets.
  • the unique label is generated in an emulsion droplet or in a series of emulsion droplets.
  • a library of uniquely-labeled agents is generated using an emulsion droplet or a series of emulsion droplets.
  • mate-pair libraries are useful for extracting distance information from sequences and are most typically used in genomic assemblies, detection of splicing in transcripts, and detection of genomic rearrangements.
  • mate-pair libraries require that DNA molecules be circularized in order to directly join the ends together (i.e., as a mate-pair).
  • the efficiency of circularization decreases as jump length increases, thus increasingly specialized techniques are required in order to prepare jumps of varying sizes.
  • the methods described herein offer a major advantage over current methodologies in that mate-pair analysis is achieved without relying on circularization and is independent of jump length, thus making it a universal mate-pair protocol potentially suitable across a range of sequencing technologies.
  • reactions are performed in emulsion droplets at single molecule dilution resulting in significant reductions in reagent costs, cycle time and input material.
  • emulsion droplets are used to segregate individual DNA molecules so that the ends of each DNA molecule can either be physically re-joined via ligation or informatically associated via analysis of the unique label.
  • the method comprises:
  • each droplet comprises a nucleic acids end-labeled on its 5' and 3' ends with oligonucleotide label, wherein the oligonucleotide label on the 5' end (the 5' oligonucleotide label) and the oligonucleotide on the 3' end (the 3' oligonucleotide label) comprise a nucleotide cohesive overhang, and wherein the nucleotide cohesive overhang on the 5' oligonucleotide label is complementary to the nucleotide cohesive overhang on the 3 ' oligonucleotide label;
  • the 5' oligonucleotide label and/or the 3' oligonucleotide comprises a biotin label.
  • the method further comprises (e) sequencing the ligated nucleic acid.
  • the DNA fragmenting agent is Nextera.
  • the method comprises:
  • each droplet comprises a nucleic acid comprising an oligonucleotide adapter
  • each index droplet comprising a first unique single-stranded detectable oligonucleotide tag, wherein the first unique single-stranded detectable oligonucleotide tag comprises a region complementary to the oligonucleotide adapter;
  • each individual library droplet comprising a melted end-labeled nucleic acid with a single index droplet from a plurality of m 2 index droplets, each index droplet comprising a second unique single-stranded detectable oligonucleotide tag, wherein the second unique single-stranded detectable oligonucleotide tag comprises a region complementary to the first unique single-stranded detectable oligonucleotide tag;
  • the method comprises:
  • each detectable oligonucleotide tags is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of a nucleic acid.
  • the 5' and 3' fragments are about 10-1000 bases (base pairs) in length, or about 10-500 bases in length, or about 10-200 bases in length.
  • the method further comprises sequencing the 5' and 3' fragments.
  • the method comprises:
  • genomic nucleic acid fragments are attached to identical unique labels at one of their ends that indicates the genomic nucleic acid fragments were separated by a known distance in a genome prior to fragmentation.
  • the pair of nucleic acid fragments were separated by greater than 10 kb in the genome prior to fragmentation. In another embodiment, the pair of nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation. In some embodiments, the method further comprises generating the pair of genomic nucleic acid fragments by fragmenting nucleic acids comprising genomic sequence and identical non- genomic sequence at their 5' and 3' ends.
  • compositions comprising:
  • paired nucleic acid fragments attached to unique labels at one end, wherein paired nucleic acid fragments:
  • paired nucleic acid fragments were separated by greater than 10 kb in the genome prior to fragmentation. In another embodiment, the paired nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation. In some embodiments, the composition is produced using any of the methods described herein.
  • nucleic acids include, but are not limited to, genomic DNA, cDNA, PCR products, mRNA, total RNA, plasmids, or fragments thereof.
  • the nucleic acids are genomic DNA, cDNA, PCR products, or fragments thereof.
  • Nucleic acids can be fragmented using methods described herein.
  • the method further comprises fragmenting uniquely end- labeled nucleic acids. Fragmenting of nucleic acids can be accomplished by methods described herein and those well-known in the art.
  • the method comprises sequencing a pair of genomic nucleic acid fragments, wherein the genomic nucleic acid fragments are attached to identical unique labels at one of their ends that indicates the genomic nucleic acid fragments were separated by a known distance in a genome prior to fragmentation.
  • the known distance is greater than 5, 10, 15, 20, 30, 40, 50, 100 kb or greater separation.
  • Genomic nucleic acid fragments can come from any organismal genomic DNA, for example, human, mammalian, bacterial, fungal or plant genomic DNA. Genomic nucleic acid fragments can be generated by fragmentation methods known in the art (see, e.g., Green and Sambrook. Molecular Cloning: A Laboratory Manual, Fourth Edition, 2012).
  • fragmentation examples include, but are not limited to, enzymatic (such as a nuclease), chemical (such as a DNA nicking agent) or mechanical (such as sonication) fragmentation.
  • Fragmentation can be random, e.g., sequence and size unspecific, or ordered, e.g., sequence dependent and/or size-restricted.
  • the fragments generated following label addition can be tailored to the limitations of the desired detection technology. For example, the fragments can be hundreds, thousands, millions or potentially billions of base pairs in length depending on the technology used to sequence the DNA.
  • Example 1 Polymer -ase-mediated bioinformatic association of nucleic acid ends for Mate-Pair Analysis
  • Genomic DNA is fragmented and size selected to a known size using techniques known in the art (e.g., sonication, cavitation, point-sink or mechanical shearing, or a DNA fragmenting enzyme and size-exclusion columns or gel purification).
  • the genomic DNA is then A-tailed and ligated to a biotinylated, T-tailed asymmetric oligonucleotide adapter using methods known in the art (see, e.g. Maniatis, Molecular Cloning). Klenow exo- enzyme is commonly used to add a single nucleotide to the 3' termini of DNA fragments).
  • the adapter is a partial duplex to allow for annealing of the single-stranded oligonucleotide indexes described below.
  • index libraries are created such that each library contains approximately >1000 unique single-stranded oligonucleotide indexes, thus approximately 2000-4000 unique indexes are used.
  • Index libraries may be created in droplets using standard flow focusing techniques. For a given library, each droplet will contain many copies of one unique single-stranded index. Droplets may contain some or all of the key components of a polymerase fill-in reaction (e.g., MgCl 2 , dNTP, and Polymerase).
  • Each unique single-stranded oligonucleotide index contains 3 distinct regions: sequence complimentary to the adapter (Ad) or to a previously added index sequence (B or C), a unique index sequence (Idx), and a sequence used to "capture” the next index oligonucleotide index which contains one or more dUTP nucleotides (B7C).
  • Fragmented genomic DNA ligated to an adapter is diluted to a desired concentration to control the number of molecules per droplet (e.g., a single DNA molecule per droplet or more than a single DNA molecule per droplet) and merged with (see above references for droplet merging) the first index library (Library "A" in Figure 2).
  • each unique single-stranded oligonucleotide index binds to the adapter on each end of the fragmented genomic DNA molecule.
  • a polymerase-mediated fill-in reaction is performed in each droplet, creating the complement to the index and capture regions on the each unique single-stranded oligonucleotide index a, and thus generating unique double- stranded oligonucleotide indexes.
  • Emulsion droplets are then broken using various mechanical or chemical reagents depending on the oil/surfactant utilized in the emulsion, resulting in the combination and mixing of the DNA from each droplet.
  • Mixed DNA is then treated with USERTM enzyme (Uracil- Specific Excision Reagent, New England BioLabs Inc., Ipswich, MA), causing the capture portion of the double-stranded oligonucleotide index to be digested due to the presence of one or more dUTP nucleotides. This digestion reveals the nascent strand, which is complementary to a sequence contained in the next library of indexes (Library "B" in Figure 2).
  • the result is fragmented genomic DNA uniquely end- labeled on both the 5' and 3' end with a unique label made up of many oligonucleotide indexes.
  • the uniquely end-labeled fragmented genomic is then fragmented and the ends are collected via streptavidin beads, which recognized the biotin label on the adapter.
  • Fragments can be ligated to technology specific sequencing adapters (e.g., Illumina adapters) and sequenced. Ends are informatically paired by matching the unique label on one fragment of DNA with the same unique label on the other fragment of DNA (see Figure 7).
  • This method of bioinformatics association can also be used with other types of nucleic acids, such as RNA, cDNA, or PCR-amplified DNA, or any other type of construct where such a labeling scheme is required.
  • Example 2 Ligation-mediated bioinformatic association of nucleic acid ends in emulsions for Mate-Pair Analysis
  • a 34 bp adapter was designed.
  • the adapter was biotinylated and T-tailed to force directionality of ligation to A-tailed lambda genomic DNA. Ligation was performed in an tube or an emulsion using 50 ng of lambda DNA and 50 ng of adapter. Lambda DNA was used as it is unlikely to form circles. Droplets were created by standard techniques (e.g., flow focusing at a T-junction using a PDMS-based microfluidic chip). Channel 1 contained DNA in ligase buffer (500 microliters) and channel 2 contained Quick Ligase in ligase buffer (500 microliters).
  • PCR primers were designed to amplify internally within the lambda DNA (ligation-independent) or to amplify a portion of the adapter and the 5' or 3' end of the lambda DNA (ligation-dependent). Negative controls were performed in tubes to ensure ligation was ligase-dependent.
  • Figure 3 shows that ligation was achieved in both tubes and emulsion droplets.
  • the forward primer for the adapter and the 5' primer for the lambda DNA only amplified in the presence of ligase, indicating that the adapter and the 5' end of the lambda DNA had ligated together in both tubes and emulsion droplets.
  • the same result was achieved using the reverse primer for the adapter and the 3' primer for the lambda DNA, indicating that the adapter and the 3' end of the lambda DNA had ligated together in both tubes and emulsion droplets.
  • Genomic DNA is fragmented and size selected to a known size using techniques known in the art as described in Example 1. The genomic DNA is then A-tailed and ligated to a biotinylated, T-tailed asymmetric oligonucleotide adapter using methods well known in the art as described in Example 1.
  • Droplet libraries (preferably 2-4 libraries) are created such that each library contains approximately 1000 unique double-stranded oligonucleotide indexes, thus approximately 2000-4000 unique indexes are used. For a given library, each droplet will contain many copies of one unique double-stranded index. Droplets may contain some or all of the key components of a ligation reaction (e.g., MgCl 2 , ATP, Ligase).
  • a ligation reaction e.g., MgCl 2 , ATP, Ligase
  • Fragmented genomic DNA ligated to an adapter is diluted to a desired concentration to control the number of molecules per droplet (e.g., a single DNA molecule per droplet or more than a single DNA molecule per droplet) and merged with the first index droplet library (Droplet Library "A" in Figure 4).
  • a desired concentration e.g., a single DNA molecule per droplet or more than a single DNA molecule per droplet
  • a ligation reaction is performed in each droplet, joining each unique double-stranded oligonucleotide index to the adapter on each end of the genomic DNA.
  • the emulsion is then broken and the DNA is phosphorylated so that a second index can be ligated to the end of the first index.
  • the result is fragmented genomic DNA uniquely end- labeled on both the 5' and 3' end with a unique label made up of many oligonucleotide indexes.
  • the uniquely end-labeled fragmented genomic is then further fragmented and the ends are collected via streptavidin beads, which recognized the biotin label on the adapter.
  • Fragments can be ligated to technology specific sequencing adapters (e.g., Illumina adapters) and sequenced. Ends are informatically paired by matching the unique label on one fragment of DNA with the same unique label on the other fragment of DNA as described in Example 1.
  • this method can be used for other types of nucleic acids, such as RNA, cDNA, or PC -amplified DNA, or any other type of construct where such a labeling scheme is required
  • the DNA was phosphorylated so that a second index could ligated to the end of the first index (two rounds of index ligation) or a third index could be ligated to the end of a second index (three rounds of index ligation).
  • a second index could be ligated to the end of the first index (two rounds of index ligation) or a third index could be ligated to the end of a second index (three rounds of index ligation).
  • the same library/pool of indexes was used (pool A).
  • a library/pool of different indexes was used (pool B).
  • Illumina indexed adapters were ligated to all three genomic DNA libraries. Libraries were then pooled and sequence on an Illumina MiSeq (Illumina, San Diego, CA) using standard Illumina sequencing primers. Paired reads were identified and analyzed en masse (i.e.
  • Figures 13 and 14 depict the results of the total read population analysis ⁇ en masse analysis) of the index ligation method.
  • Library 1 which underwent 1 round of index ligation, had an expected outcome of an index read in position 1 and an adapter read in position 2.
  • Figure 15 depicts the results of read pair analysis of individual molecules that underwent the index ligation method.
  • reads were paired so that a molecule-by-molecule analysis was performed.
  • reads were paired based on their unique read identifier. Each read was then broken down into 4 positions (8-mers) per read as described above.
  • Figure 5 Figure 5
  • Figure N The composition of the top 10 most prevalent molecular outcomes and the number of pairs for each outcome are also shown in Figure 5 ( Figure N). It was determined that the most desired outcome (the correct expected outcome) occurred 6% of the time in Library 1, 4% of the time in Library 2, and 4% of the time in Library 3.
  • Figures 13-15 show that the expected outcome was achieved and thus index ligation was a valid method of generating a unique label.
  • DNA samples are sheared to a desired size then the "Cap” and random combinations of index sequences are symmetrically attached to the fragment ends via ligation.
  • a new adapter containing an Illumina sequencing primer (SP1) adjacent to the Illumina P7 sequence is attached to the ends of the molecules via ligation as described above.
  • the population of molecules is then incubated in the presence of a transposome carrying a different Illumina sequencing primer (SP2) adjacent to the Illumina P5 sequence. This reaction creates many fragments where both ends are flanked by the Illumina P5 sequence, but only two fragments per molecule that carry both the Illumina P7 and P5 sequences.
  • PCR amplification using primers to P5/P7 is performed in order to enrich/select the fragment ends. Enrichment of Ends via/rc Vitro Transcription
  • a primer containing a random nucleotide sequence of a set length (i.e., pentamer, hexamer, etc.) flanked by a different Illumina sequencing primer (SP2) is utilized as the primer in a reverse transcription reaction.
  • RNA molecules may be trimmed to a desired size range and ligated to the Illumina sequencing primer (SP2) via standard techniques.
  • Illumina P5 and P7 sites are then added to the cDNA via PCR using primers carrying Illumina P5-SP1 and P7-SP2 sequences.
  • DNA samples are sheared to a desired size then the Cap and random combinations of index sequences are symmetrically attached to the fragment ends via ligation.
  • a new adapter containing an Illumina sequencing primer (SP1) adjacent to the Illumina P7 sequence is attached to the ends of the molecules via ligation as described above.
  • the population of molecules is then incubated in the presence of Fragmentase or a cocktail of restriction endonucleases to liberate the ends of the molecules. Fragments are then tailed at the 3' end using terminal transferase to attach a set number of specific nucleotides to the fragment ends, effectively creating a common priming sequence on the ends of all molecules.
  • priming sequences may be ligated to the 3' of the molecules using standard techniques.
  • the fragments are then amplified via PCR using SP2-P7 and SP1-P5 primers where the SP1-P5 primer contains a tail complementary to the priming site attached in the previous step.
  • a method for labeling a nucleic acid at both its 5 ' and 3 ' ends with a unique label comprising the steps of:
  • each detectable oligonucleotide tag is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of said nucleic acid,
  • each end-labeled nucleic acid is identically labeled at its 5 ' and 3 ' ends.
  • n 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 , 10 3 , 10 4 , 10 5 , or 10 6 or more detectable oligonucleotide tags.
  • a method comprising:
  • each detectable oligonucleotide tag is randomly selected from a plurality of detectable oligonucleotide tags, thereby generating a second nucleic acid comprising the first nucleic acid attached at its 5' and/or 3' end with a unique combination of detectable oligonucleotide tags, wherein the plurality of second nucleic acids is generated using emulsion droplets.
  • the second nucleic acid is a genomic DNA fragment attached to the unique combination of detectable oligonucleotide tags at its 5' or 3' end.
  • the second nucleic acid is a genomic DNA fragment attached to the same unique combination of detectable oligonucleotide tags at its 5' and 3' end.
  • a method comprising:
  • each index droplet comprising a plurality of one unique detectable oligonucleotide tag
  • step (f) amplifying the end-labeled nucleic acid formed in step (e).
  • end-labeling comprises ligation of the unique oligonucleotide tag with the nucleic acid.
  • end-labeling comprises a polymerase-mediated fill-in reaction.
  • polymerase-mediated fill-in reaction comprises:
  • a labeled nucleic acid obtainable by the method of paragraph 1.
  • amplification step (f) comprises the steps of:
  • amplification step (f) comprises the steps of:
  • amplification step (f) comprises the steps of:
  • step (iv) performing PCR amplification on the fragments formed in step (iii) using a second sequencing primer.

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Abstract

The invention provides for methods for uniquely labeling populations of nucleic acids of interest in emulsion droplets using random combinations of oligonucleotides. The labeling methodology of the invention may be used, inter alia, to generate mate pair genomic fragments without the need for circularization. Because the method is independent of circularization, mate pairs can be generated from any length genomic fragment.

Description

COMPOSITIONS AND METHODS FOR LONG INSERT, PAIRED END LIBRARIES OF NUCLEIC ACIDS IN EMULSION DROPLETS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Application Serial No. 61/779,964, filed March 13, 2013; 61/731,021, filed November 29, 2012; and 61/703,884, filed September 21, 2012. This application is related to U.S. Provisional Application Serial No. 61/779,999, filed March 13, 2013.
[0002] The foregoing application, and all documents cited therein ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
FIELD OF THE INVENTION
[0003] The invention is directed to methods for uniquely labeling populations of nucleic acids of interest in emulsion droplets using random combinations of oligonucleotides.
BACKGROUND OF INVENTION
[0004] The ability to label and create mate-pair libraries serves multiple purposes in research and industry. However, several limitations exist in current labeling technologies. These limitations include the limited number of distinguishable detectable moieties currently in existence, the amount of time required to uniquely label a plurality of library elements, the requirement, in some instances, for specialized equipment, and the cost involved.
SUMMARY OF INVENTION
[0005] The invention provides methods and compositions for uniquely labeling nucleic acids, such as DNA, peptides or proteins. One of the major limitations of prior art labeling techniques is the limited number of available unique labels. Typically the number of nucleic acids to be labeled in any given application far exceeds the number of unique labels that are available. The methods of the invention can be used to synthesize essentially an infinite number of unique labels. Moreover, because of their nature, the labels can be easily detected and distinguished from each other, making them suitable for many applications and uses. The methods of the invention also provides for amplifying nucleic acids to increase the number of read pairs properly mated via their unique index combination. Additionally, the methods of the invention allow for each end-labeled nucleic acid to be identically labeled at its 5' and 3' ends.
[0006] In one embodiment, provided is a method for labeling a nucleic acid at both its 5 ' and 3' ends with a unique label, comprising the steps of:
a) providing a pool of nucleic acids; and
b) sequentially end-labeling said nucleic acids with a random combination of n detectable oligonucleotide tags, each of said oligonucleotide tags optionally comprising a cohesive overhang of x base pairs in length, wherein each detectable oligonucleotide tag is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of said nucleic acid,
wherein said method is performed in emulsion droplets, and wherein each end-labeled nucleic acid is identically labeled at its 5' and 3' ends.
[0007] Also provided are labeled nucleic acids and libraries of said nucleic acids.
BRIEF DESCRIPTION OF DRAWINGS
[0008] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying Figures, which are schematic and are not intended to be drawn to scale. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.
[0009] Figure 1 shows that the efficiency of DNA circularization (cyclization) decreases as fragment length increases.
[0010] Figure 2 is a schematic depicting the technique for polymerase mediated index addition. [0011] Figure 3 is a gel electrophoresis showing ligation of an adapter sequence in either a tube (T) or an emulsion droplet (E).
[0012] Figure 4 is a schematic depicting the technique for ligation mediated index addition.
[0013] Figure 5 is a schematic depicting the technique for symmetric ligation-mediated index addition.
[0014] Figure 6 is a flowchart detailing the criteria for barcode sequence selection.
[0015] Figure 7 is a schematic depicting the methodology for informatically deriving mate pairs.
[0016] Figure 8 shows catalyzing ligation by the controlled addition of MgCl2.
[0017] Figure 9 shows a stability determination of MgCl2 in droplets.
[0018] Figure 10 shows a determination of the optimal ratio of genomic Index:genomic DNA.
[0019] Figure 11 is a schematic depicting the process of symmetric indexing in emulsion.
[0020] Figure 12 shows a proof of concept experiment.
[0021] Figure 13 shows an analysis of E. coli proof of concept libraries.
[0022] Figure 14 shows an analysis of lambda proof of concept libraries.
[0023] Figure 15 shows a determination of symmetry of indexing in E. coli proof of concept libraries.
[0024] Figure 16 is a schematic of a mate pair synthesis process using single stranded genomic DNA as the agent.
[0025] Figure 17 is a schematic of a mate pair synthesis using droplets and Nextera transposomes as detectable tags.
[0026] Figure 18 shows the determination of uniformity of blunt-ended indexing.
[0027] Figure 19 shows impact of ligation efficiency on bioinformatics end association.
[0028] Figure 20 shows a redesign of index sequences.
[0029] Figure 21 shows uniformity of indexing.
[0030] Figure 22 shows amplification of fragment ends via transposome-based selection.
[0031] Figures 23a and 23b illustrate enrichment of ends via in vitro transcription.
[0032] Figure 24 shows amplification of ends via anchored PCR. DETAILED DESCRIPTION OF INVENTION
[0033] The methods of the invention easily and efficiently generate libraries of unique labels. Such libraries may be of any size, and are preferably large libraries including hundreds of thousands to billions of unique labels. The libraries of unique labels may be synthesized separately or may be synthesized in real-time (e.g., while in the presence of the nucleic acid). Methods for nucleic acid sequencing and detection of non-nucleic acid detectable moieties are known in the art and are described herein.
[0034] The methods of the invention label nucleic acids in emulsion droplets. The nucleic acid is identically labeled at its 5' and 3' ends. Further, the nucleic acids are amplified by a method such as, for example, anchored PCR.
[0035] The invention further provides for methods for creating mate-pair libraries of uniquely labeled nucleic acids such as genomic DNA fragments. The term mate-pair, although specific to certain next generation sequencing technologies, is intended to generically describe a "jumping" library. A jumping library is any DNA construct where the physical genomic distance between sequencing reads can be derived without the need to sequence the entire intervening length of DNA. Depending on the application and/or sequencing technology, some terms frequently used to describe such libraries include (but are not limited to): jumping libraries, distance libraries, long range libraries, linking libraries, long distance linking libraries, mate-pair libraries and long paired-end libraries.
[0036] The invention contemplates that the labels are prepared by sequentially attaching randomly selected oligonucleotides (referred to interchangeably as oligonucleotide tags) to each other. The order in which the oligonucleotides attach to each other is random and in this way the resultant label is unique from other labels so generated. The invention is based, in part, on the appreciation by the inventors that a limited number of oligonucleotides can be used to generate a much larger number of unique labels. The invention therefore allows a large number of labels to be generated (and thus a large number of nucleic acids to be uniquely labeled) using a relatively small number of oligonucleotides.
[0037] The unique labeling strategies of the invention provide methods for generating mate pairs from genomic DNA fragments of virtually any length. This is a significant advantage over the mate pair methods of the prior art which require circularization of the genomic DNA fragment and thus are limited by the length of the fragment. In contrast, the methods of the invention do not rely on circularization of the genomic DNA fragments and thus are able to generate mate pairs from genomic DNA fragments of various lengths.
[0038] Certain aspects of the invention, and the advantages of the invention over previous techniques, are shown in the Figures. Figure 1 shows that the efficiency of DNA circularization (cyclization) decreases as fragment length increases. A) Predicted probability of circularization for DNA fragments ranging from lkb to lOOkb. Probability values are derived using the Jacobson-Stockmayer factor, j=(3/2pbl)3/2 (where l=contour length and b=hydrodynamic segment length), which is a probability density function that describes the effective concentration of one end in the neighborhood of the other end on the same molecule of a random coil polymer. B) Portion of the graph presented in panel A focused on the lkb to 12kb size range. C) Illumina mate-pair libraries were prepared from human genomic DNA fragments of the sizes indicated. Estimated jumping library complexity (shown on the Y-axis) decreases rapidly as fragment size increases due largely to inefficiencies at circularization.
[0039] Figure 2 shows the technique for polymerase mediated index addition. As seen in Figure 2, (1) genomic DNA is size selected, end repaired and A-tailed. (2) The biotinyalted adapter can only ligate in one orientation on each end of the genomic DNA due to the presence of the T-tail. The adapter is a partial duplex to allow for primer annealing in the next step of the process. (3) Multiple index libraries are created in emulsion and a polymerase-driven fill-in reaction is used to add the index. The index libraries may contain some or all of the key components of the fill in reaction (i.e., MgCl2, dNTP, polymerase). The library is comprised of unique single stranded DNA oligonucleotides (oligos). Each oligo will contain 3 distinct moieties: sequence complimentary to adapter (Ad), a unique index sequence (Idx) and a sequence used to "capture" the next index oligo which contains one or more dUTP nucleotides (B'/C). DNA is diluted to a desired concentration to control the number of molecules per droplet and is merged with the index droplet library. (4) A fill-in reaction is performed creating the complement to the index and the "capture" site. (5) The emulsion is broken and DNA is pooled prior to treatment with USER enzyme causing the "capture" portion of the indexed oligo to be digested/released from the nascent strand which does NOT contain dUTP. (6-8) The process of DNA dilution, index addition, fill-in, pool, and USER enzyme treatment is repeated for the desired number of cycles, each time adding one new index to the end of the DNA. (9) After the final index addition, DNA is fragmented and the ends are collected via streptavidin beads. All fragments are ligated to technology specific sequencing adapters and ends are informatically paired based on their unique string of indexes.
[0040] Figure 3 is a gel electrophoresis showing ligation of an adapter sequence in either a tube (T) or an emulsion droplet (E).
[0041] Figure 4 is a schematic of the technique for ligation mediated index addition. As shown in Figure 4, (1) Genomic DNA is size selected, end repaired and A-tailed. (2) The biotinyalted adapter can only ligate in one orientation on each end of the genomic DNA due to the presence of the T-tail. The adapter may be blunt ended or it may be a partial duplex with a sequence specific cohesive overhang to allow for index annealing in the next step of the process. (3) Multiple index libraries are created in emulsion and a ligation reaction is performed to add the index. The index libraries may contain some or all of the key components of the ligation reaction (i.e., MgCl2, dNTP, ligase). Each droplet will contain many copies of an individual (unique) index sequence. DNA is diluted to a desired concentration to control the number of genomic fragments per droplet and is then joined to the index library allowing the ligation reaction to occur. Following ligation, the emulsion is broken and DNA is pooled, purified and prepared so that it can accept the next index. (4-6) The process of DNA dilution, index addition, ligation, pooling, clean-up and phosphorylation is repeated for the desired number of cycles, each time adding one new index to the end of the DNA. (7) After the final index addition, DNA is fragmented and the ends are collected via streptavidin beads. All fragments are ligated to technology specific sequencing adapters and ends are informatically paired based on their unique string of indexes.
[0042] Figure 5 depicts the technique for symmetric ligation-mediated index addition. (1) Genomic DNA is size selected, end repaired and A-tailed. (2) The biotinyalted adapter can only ligate in one orientation on each end of the genomic DNA due to the presence of the T-tail. The adapter may be blunt ended or it may be a partial duplex with a sequence specific cohesive overhang to allow for index annealing in the next step of the process. (3) Multiple index libraries are created and a ligation reaction is performed to add the index. Following ligation, DNA molecules are pooled, purified and prepared for the next round of ligation. (4) This process is repeated Y number of times. The total "diversity" of the population of unique index combinations is dictated by the number of indexes used raised to the power of the number of cycles performed. For example, 3 round of index ligation using a 1152 element array creates 1152 or 1,528,823,808 combinations. (5) After the final index addition, DNA is fragmented and the ends are collected via streptavidin beads. (6) Sheared DNA fragments are end repaired as needed and ligated to technology specific sequencing adapters. Following sequencing, the ends are informatically paired based on their unique string of indexes.
[0043] Figure 6 is a flowchart detailing the criteria for barcode sequence selection.
[0044] Though the set started with a hypothetical pool of >1 million random sequences, the filtering criteria eliminated over 99% of the random sequences to arrive at a collection of ~5,000 barcode sequences.
[0045] Figure 7 provides the methodology for informatically deriving mate pairs. Left Panel: For a standard Ilumina mate-pair library, each construct is sequenced using two reads. Since both ends of the parent genomic DNA fragment are contained in a single construct, two reads (i.e., a single read pair) are sufficient to establish a mate-pair. Right Panel: Unlike a standard library, a symmetrically indexed library requires a total of 4 reads (i.e., 2 read pairs) in order to establish a mate-pair. For a given construct, one read will contain the index information while the other is genomic DNA (i.e, one read pair defines one "half of the mate-pair). For each construct in the library, the algorithm must search the data set to identify appropriately matching indexing combinations. Once the index combinations are matched, their corresponding genomic reads can be positioned relative to the genome.
[0046] Figure 8 shows catalyzing ligation by the controlled addition of MgCl2. In order to prevent spontaneous ligation (concatamerization) of genomic DNA fragments, it is necessary to prepare the DNA in a solution that lacks one or more of the key components necessary to catalyze a ligation reaction. Due to stability and cost issues, sequestration of MgCl2 in the index droplets would be preferable over other key components (i.e., ligase enzyme and ATP). A single 380bp restriction fragment was generated from pBR322 plasmid DNA and prepared it such that it can only ligate to itself in one orientation. Thus, a 760bp ligated product is easily distinguishable from the unligated 380bp product. A 10X modified ligation buffer lacking MgCl2 was prepared but containing 500mM Tris-HCl pH 7.5, lOOmM dithiothreitol and lOmM ATP. For lanes 1-3 and lanes 7 and 8, reactions were prepared as indicated, incubated for 1 hour at room temperature, purified and run on an Agilent DNA 1000 chip. For lanes 4, 5 and 6, reactions were prepared as indicated and incubated for 1 hour at room temperature. Following this initial incubation, either water (lane 4), MgCl2 (lane 5) or T4 DNA ligase (lane 6) were added to the reaction. Samples were then allowed to incubate for an additional 1 hour at room temperature before being purified and run on an Agilent DNA 1000 chip. No ligated product was observed in those reactions lacking MgCl2 (lanes 1 and 4). Upon addition of 50mM MgCl2 at either time = 0 (lane 2) or time = 1 hour (lane 5), ligated product was observed.
[0047] Figure 9 shows stability determination of MgCl2 in droplets. Droplets containing 50mM, lOmM or ImM concentrations of MgCl2 were prepared and stored at +4°C for ~4.5 days. Droplets were then broken and the aqueous phase was collected from each droplet "library" and transferred into fresh 1.5ml tubes. Ligation reactions containing IX Modified Ligation buffer (i.e., buffer lacking MgCl2), T4 DNA ligase and 380bp control fragment DNA were prepared. Aqueous phase recovered from the various droplet libraries (lanes 1-3) or non-emulsified MgCl2 (lanes 4-6) was added to the various ligation reactions. The 50mM and lOmM reactions appeared to perform equally well while a marked reduction in the amount of ligated product was observed for the ImM condition. Importantly, the MgCl2 released from the droplet library (lanes 1 -3) appeared equally capable of catalyzing the ligation reaction as freshly added MgCl2 (lanes 4-6). NOTE: The 50mM droplet condition looks slightly less intense on the gel image due to slightly less material being loaded on the gel for that lane.
[0048] Figure 10 represents a determination of the optimal ratio of genomic Index: genomic DNA. Lambda genomic DNA was sheared to a mean size of ~300bp using a Covaris S2 instrument. The genomic DNA was then end repaired and utilized in a ligation reaction containing variable molar ratios of Index:gDNA. Following index ligation, samples were end repaired, A-tailed and ligated to Illumina adapters. Samples were pooled and sequenced on an Illumina MiSeq. The percentage of reads where indexed was observed is shown. NOTE: The indexes used in this experiment were blunt ended 20bp sequences.
[0049] Figure 11 provides the process of symmetric indexing in emulsion. Index libraries are prepared in an emulsion. The droplets carrying index also contain a concentration of MgCl2 such that when they are joined with a solution of DNA, ligase buffer and ligase enzyme, the final concentration of MgCl2 in a given reaction is 50mM. Although many molecules/particles receive the same index in any given round of addition, the probability that any two identically indexed molecules/particles will travel together in a subsequent round of index addition is extremely low. After each round of index addition, the emulsion is broken and DNA samples are purified and prepared for the next round of index addition. [0050] Figure 12 shows a proof of concept experiment. E. coli genomic DNA was sheared to a mean size of approximately 300bp using a Covaris S2 instrument, end repaired, A-tailed and ligated to the cap adapter. Lambda genomic DNA was prepared similarly, but was not sheared. Genomic DNA fragments were then subjected to 1, 2 or 3 rounds of blunt-ended index ligation in bulk (i.e., in microcentrifuge tubes). E. coli fragments were not sheared following index ligation while lambda fragments were sheared to approximately 500bp using a Covaris S2 instrument. Cap containing fragments were selected via incubation with paramagnetic streptavidin M-280 beads, end repaired, A-tailed and ligated to Illumina sequencing adapters. Samples were pooled and sequenced on an Illumina MiSeq using standard paired end chemistry.
[0051] Figure 13 is an analysis of E. coli proof of concept libraries. E. coli genomic DNA libraries were prepared in duplicate (Condi and Cond2) as described above (see Figure 12). Libraries were pooled and sequenced with a lOlbp paired read on a single MiSeq run. Paired reads that passed filter were analyzed together as a single population. (A) Reads were broken down into 20bp units (i.e., positions) and checked for the presence of index sequences. The expected outcome at a given position is shown (Idx = index, Ad = capping adapter, E = E. coli genomic DNA). (B) The number of reads containing index at a given position are shown. (C) The percent of reads containing index at a given position is shown.
[0052] Figure 14 provides an analysis of lambda proof of concept libraries. Lambda phage genomic DNA libraries were prepared in duplicate (Condi and Cond2) as described above (see Figure 12). Libraries were pooled and sequenced with a lOlbp paired read on a single MiSeq run. Paired reads that passed filter were analyzed together as a single population. (A) Reads were broken down into 20bp units (i.e., positions) and checked for the presence of index sequences. The expected outcome at a given position is shown (Idx = index, Ad = capping adapter, L = lambda genomic DNA). (B) The number of reads containing index at a given position are shown. (C) The percent of reads containing index at a given position is shown. The percentages shown are corrected for the fact that one half of the reads will necessarily be the genomic "end" of the library insert.
[0053] Figure 15 shows a determination of symmetry of indexing in E. coli proof of concept libraries. E. coli genomic DNA was prepared as described above (see Figure 12). Libraries subjected to 1 (panels 1A and IB), 2 (panels 2A and 2B) or 3 (panels 3A and 3B) rounds of index ligation are shown. All data that passed filter was analyzed as read pairs which were then broken down into 20bp units (i.e., positions) and checked for the presence of index sequences. Positions where index sequences were detected are depicted by green boxes; positions where indexes were not detected are depicted by white boxes. The expected outcome for each library is denoted by a green asterisk.
[0054] Figure 16 is a schematic representation of a mate pair synthesis process using single stranded genomic DNA as the agent. Each droplet comprises both strands of the genomic fragment. As shown in the Figure, the strands are identically labeled at one end.
[0055] Figure 17 is a schematic of a mate pair synthesis using droplets and Nextera transposomes as detectable tags.
[0056] Figure 18 shows the determination of uniformity of blunt-ended indexing. C57BL/6J mouse genomic DNA was sheared to approximately 40kb using a Genemachines Hydroshear. Samples were run on a 0.7% agarose gel and fragments of approximately 31kb and 38kb were collected and purified separately. All fragments were then end repaired, A-tailed and ligated to the biotinylated cap adapter. Fragments were then ligated to blunt-ended index sequences contained in a droplet library using the Raindance Thunderstorm instrument. Following each round of index ligation, the emulsion was broken and samples were end repaired and purified for use in subsequent rounds of ligation. A total of 3 rounds of index ligation were performed. After the final round of index ligation, samples were sheared to ~500bp in length using a Covaris S2 instrument. Fragments containing the biotinylated cap adapter were selected using streptavidin M-280 beads, end repaired, A-tailed and ligated to Illumina sequencing adapters. Samples were then sequenced using an Illumina MiSeq instrument. The location of the cap sequence within a given read was determined. The total number of reads with cap sequence identified at a given position within the read are shown. Significant populations of reads where the cap was located at bp 1 (i.e., no index present), bp 21 (1 index), bp 41 (2 indexes) and bp 61 (3 indexes) were observed for both the 3 lkb and 38kb libraries.
[0057] Figure 19 describes impact of ligation efficiency on bioinformatics end association. Upon analysis of the indexed mouse libraries (see Figure 18), it was observed that clear populations of reads carrying 1, 2 or 3 indexes were present in the final library. The fact that multiple distinct populations were observed indicates a lack of symmetry in the indexing protocol. This presents a challenge to the informatic association of mate pairs since, for example, a read carrying 3 indexes may have its mate in the pool of reads carrying 3, 2 or 1 indexes. Thus, the likelihood of correctly pairing reads decreases as the number of indexes present decreases.
[0058] Figure 20 is a redesign of index sequences to improve ligation efficiency. The cap adapter and all barcodes were redesigned to carry a 4-bp cohesive overhang on either side of the barcode (but only on one side of the cap adapter). Barcodes were separated into 4 different populations (A, B, C and D) depending on the sequence of the 4-bp cohesive overhang. The sequence of the cohesive overhang for each population is shown.
[0059] Figure 21 shows uniformity of indexing using cohesive overhang indexing. E. coli and lambda genomic DNA libraries were prepared as described above (see Figure 12), but this time using cohesive overhang indexes in conjunction with a cohesive overhang ended cap adapter. Reads were analyzed as before and the location of the biotinylated cap within the read was determined. The total number of reads with cap sequence identified at a given position within the read are shown. This analysis revealed a clear improvement in the homogeneity of the indexed read population where the vast majority of the reads from the cohesive overhang indexed libraries carried 3 indexes
[0060] Figures 22 to 24 show examples of fragment amplification techniques. Figure 22 shows transposome-based selection and amplification of ends creating many fragments where both ends are flanked by an Illumina P5 sequence. Figure 23 shows enrichment of ends via in vitro transcription wherein T7 RNA polymerase is used in order to amplify both ends of a given molecule. Figure 24 shows amplification using an anchored PCR technique described in Example 3.
[0061] Thus, in one aspect, the invention provides a method comprising generating a plurality of unique labels by attaching at least two, randomly selected, detectable oligonucleotide tags to each other in a sequential manner, and associating each unique label with a separate nucleic acid.
[0062] In some embodiments, the at least two detectable tags are attached to each other using ligation, polymerization, or a combination thereof.
[0063] In some embodiments, the unique label is generated in an emulsion droplet or in a series of emulsion droplets. In some embodiments, the library of uniquely-labeled nucleic acids is generated using an emulsion droplet or a series of emulsion droplets. [0064] In another aspect, the invention provides a method comprising sequentially attaching at least two detectable oligonucleotide tags to a 5' and/or 3' end of a first nucleic acid, wherein each detectable oligonucleotide tag is randomly selected from a plurality of detectable oligonucleotide tags, thereby generating a second nucleic acid comprising the first nucleic acid attached at its 5' and/or 3' end with a unique combination of detectable oligonucleotide tags.
[0065] In some embodiments, the first nucleic acid is a genomic DNA fragment.
[0066] In another aspect method comprising sequentially end-labeling nucleic acids in a plurality, at their 5' and 3' ends, with a random combination of n detectable oligonucleotide tags, wherein each end-labeled nucleic acid is (a) identically labeled at its 5' and 3' ends, and (b) uniquely labeled relative to other nucleic acids in the plurality, wherein each detectable oligonucleotide tags is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of a nucleic acid.
[0067] In some embodiments, the number of oligonucleotides is 10-fold, 100-fold, 1000- fold, or 10000-fold less than the number of nucleic acids.
[0068] In some embodiments, the method further comprises fragmenting end-labeled nucleic acids into at least a 5' fragment comprising the 5' end of the nucleic acid attached to the random combination of n oligonucleotide tags and into a 3' fragment comprising the 3' end of the nucleic acid attached to the random combination of n oligonucleotide tags.
[0069] In some embodiments, the 5' and 3' fragments are about 10-1000 bases (base pairs) in length, or about 10-500 bases in length, or about 10-200 bases in length. In some embodiments, the method further comprises sequencing the 5' and 3' fragments.
[0070] In another aspect, the invention provides a method comprising (a) end-labeling two or more first subsets of nucleic acids with a detectable oligonucleotide tag to produce nucleic acids within a subset that are identically end-labeled relative to each other and uniquely end-labeled relative to nucleic acids in other subsets; (b) combining two or more subsets of uniquely end- labeled nucleic acids to form a pool of nucleic acids, wherein the pool comprises two or more second subsets of nucleic acids that are distinct from the two or more first subsets of nucleic acids; (c) identically end-labeling two or more second subsets of nucleic acids with a second detectable oligonucleotide tag to produce nucleic acids within a second subset that are uniquely labeled relative to nucleic acids in the same or different second subsets; and (d) repeating steps (b) and (c) until a number of unique end-label combinations is generated that exceeds the number of starting nucleic acids.
[0071] In another aspect, the invention provides a method comprising (a) providing a pool of nucleic acids; (b) separating the pool of nucleic acids into sub-pools of nucleic acids; (c) end- labeling nucleic acids in each sub-pool of with one of mj detectable oligonucleotide tags thereby producing sub-pools of labeled nucleic acids, wherein nucleic acids in a sub-pool are identically end-labeled to each other; (d) combining sub-pools of labeled nucleic acids to create a pool of labeled nucleic acids; (e) separating the pool of labeled nucleic acid molecules into second sub- pools of labeled nucleic acids; (f) repeating steps (c) to (e) n times to produce nucleic acids end- labeled with n detectable oligonucleotide tags wherein the pool in (a) consists of a number of nucleic acids that is less than (mi)(m2)(m3) ... (m„).
[0072] In another aspect, the invention provides a method comprising (a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acid; (b) fusing each individual library droplet with a single index droplet from a plurality of mi index droplets, each index droplet comprising a plurality of one unique detectable oligonucleotide tag; (c) end-labeling the nucleic acid with the unique detectable oligonucleotide tag in a fused droplet; (d) harvesting end-labeled nucleic acids from the fused droplets and generating another population of library droplets comprising end-labeled nucleic acids; and (e) repeating steps (b) to (d) n times to produce nucleic acids end-labeled with n unique detectable oligonucleotide tag, wherein the n unique detectable oligonucleotide tags generate an (mi)(m2)(m3)...(mn) number of combinations that is greater than the number of starting nucleic acids.
[0073] In some embodiments, end-labeling comprises ligation of the unique oligonucleotide tag with the nucleic acid. In some embodiments, the unique oligonucleotide tag is double- stranded. In some embodiments, the method further comprises phosphorylating the nucleic acids between steps (b) and (c).
[0074] In some embodiments, end-labeling comprises a polymerase-mediated fill-in reaction. In some embodiments, the polymerase-mediated fill-in reaction comprises (a) producing a single-stranded cohesive overhang on the nucleic acid, wherein the cohesive overhang is complementary to one end of the unique detectable oligonucleotide tag; (b) annealing the complementary end of the unique oligonucleotide tag to the single-stranded cohesive overhang such that at least one nucleotide of the unique detectable oligonucleotide tag is not annealed to the nucleic acid, producing a unique detectable oligonucleotide tag cohesive overhang; and (c) extending the single-stranded cohesive overhang of (a) using a polymerase and nucleotides complementary to the unique detectable oligonucleotide tag cohesive overhang to produce a double-stranded unique detectable oligonucleotide tag.
[0075] In some embodiments, the single-stranded cohesive overhang on the nucleic acid is produced by a USER enzyme. In some embodiments, the unique detectable oligonucleotide tag is single-stranded.
[0076] In some embodiments, an oligonucleotide adapter is added to the nucleic acids before labeling with the unique detectable oligonucleotide tags. In some embodiments, the adapter comprises biotin. In some embodiments, the adapter comprises a thymidine tail cohesive overhang.
[0077] In some embodiments, labeling occurs at the 5' and 3' ends of the nucleic acid. In some embodiments, labeling occurs at the 5' or the 3' end of the nucleic acid.
[0078] In some embodiments, the nucleic acids are genomic DNA, cDNA, PCR products, or fragments thereof.
[0079] In some embodiments, the method further comprises fragmenting uniquely end- labeled nucleic acids. In some embodiments, the method further comprises sequencing the uniquely end-labeled nucleic acids.
[0080] In some embodiments, the number of nucleic acids in the pool is at least two times greater than the number of unique oligonucleotide tags.
[0081] In another aspect, the invention provides a method comprising (a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acids end-labeled on its 5' and 3' ends with oligonucleotide label, wherein the oligonucleotide label on the 5' end (the 5' oligonucleotide label) and the oligonucleotide on the 3' end (the 3' oligonucleotide label) comprise a nucleotide cohesive overhang, and wherein the nucleotide cohesive overhang on the 5' oligonucleotide label is complementary to the nucleotide cohesive overhang on the 3' oligonucleotide label; (b) fusing each individual library droplet with a droplet comprising a DNA fragmenting enzyme, thereby producing a fused droplet; (c) fragmenting the nucleic acid with the 5' and 3' oligonucleotide labels in the fused droplet, thereby producing a fused droplet comprising a nucleic acid fragment comprising the 5' oligonucleotide label and a nucleic acid fragment comprising the 3' oligonucleotide label; and (d) ligating the 5' oligonucleotide label and the 3' oligonucleotide label nucleic acid, thereby ligating the nucleic acid fragment comprising the 5' oligonucleotide label and the nucleic acid fragment comprising the 3' oligonucleotide label, thereby producing a ligated nucleic acid.
[0082] In some embodiments, the 5' oligonucleotide label and/or the 3' oligonucleotide comprises a biotin label.
[0083] In some embodiments, the method further comprises (e) sequencing the ligated nucleic acid.
[0084] In some embodiments, the DNA fragmenting agent is Nextera.
[0085] In another aspect, the invention provides a method comprising (a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acid comprising an oligonucleotide adapter; (b) melting the nucleic acid; (c) fusing each individual library droplet comprising a melted nucleic acid with a single index droplet from a plurality of ml index droplets, each index droplet comprising a first unique single-stranded detectable oligonucleotide tag, wherein the first unique single-stranded detectable oligonucleotide tag comprises a region complementary to the oligonucleotide adapter; (d) annealing the first unique single-stranded detectable oligonucleotide tag to the nucleic acid and performing a fill-in reaction, thereby producing an end-labeled nucleic acid; (e) harvesting end- labeled nucleic acids from the fused droplets and generating another population of library droplets, wherein each droplet comprises an end-labeled nucleic acid; (f) melting the end-labeled nucleic acid; (g) fusing each individual library droplet comprising a melted end-labeled nucleic acid with a single index droplet from a plurality of m2 index droplets, each index droplet comprising a second unique single-stranded detectable oligonucleotide tag, wherein the second unique single-stranded detectable oligonucleotide tag comprises a region complementary to the first unique single-stranded detectable oligonucleotide tag; (h) annealing the secoiid unique single-stranded detectable oligonucleotide tag to the nucleic acid and performing a fill-in reaction, thereby producing an end-labeled nucleic acid; (i) harvesting end-labeled nucleic acid molecules from the fused droplets and generating another population of library droplets comprising end-labeled nucleic acids; and (j) repeating steps (f) to (i) n times to produce nucleic acids end-labeled with n detectable oligonucleotide tags, wherein the n detectable oligonucleotide tags generate an (m1)(m2)(m3)...(mn) number of combinations that is greater than the number of starting nucleic acids.
[0086] In another aspect, the invention provides a method comprising sequencing a pair of genomic nucleic acid fragments, wherein the genomic nucleic acid fragments are attached to identical unique labels at one of their ends that indicates the genomic nucleic acid fragments were separated by a known distance in a genome prior to fragmentation.
[0087] In some embodiments, the pair of nucleic acid fragments were separated by greater than 10 kb in the genome prior to fragmentation.
[0088] In some embodiments, the pair of nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation.
[0089] In some embodiments, the method further comprises generating the pair of genomic nucleic acid fragments by fragmenting nucleic acids comprising genomic sequence and identical non-genomic sequence at their 5' and 3' ends.
[0090] In another aspect, the invention provides a composition comprising a plurality of paired nucleic acid fragments attached to unique labels at one end, wherein paired nucleic acid fragments:
[0091] (a) share an identical unique label at one end that is unique in the plurality, and (b) were separated from each other in a genome by a known distance prior to fragmentation.
[0092] In some embodiments, paired nucleic acid fragments were separated by greater than
10 kb in the genome prior to fragmentation. In some embodiments, paired nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation.
[0093] In another aspect, the invention provides a composition comprising a plurality of paired genomic nucleic acid fragments produced any of the foregoing methods.
[0094] The present invention further encompasses methods of making and/or using one or more of the embodiments described herein.
[0095] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying Figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.
[0096] As used herein, "agent" or "agents" refers to a nucleic acid. The nucleic acid agent may be single-stranded (ss) or double-stranded (ds), or it may be partially single-stranded and partially double-stranded. Nucleic acid agents include but are not limited to DNA such as genomic DNA fragments, PCR and other amplification products, RNA, cDNA, and the like. Nucleic acid agents may be fragments of larger nucleic acids such as but not limited to genomic DNA fragments.
Association of Labels and Agents
[0097] An agent of interest may be associated with a unique label. As used herein, "associated" refers to a relationship between the agent and the unique label such that the unique label may be used to identify the agent, identify the source or origin of the agent, identify one or more conditions to which the agent has been exposed, etc. A label that is associated with an agent may be, for example, physical attached to the agent, either directly or indirectly, or it may be in the same defined, typically physically separate, volume as the agent. A defined volume may be an emulsion droplet, a well (of for example a multiwell plate), a tube, a container, and the like. It is to be understood that the defined volume will typically contain only one agent and the label with which it is associated, although a volume containing multiple agents with multiple copies of the label is also contemplated depending on the application.
[0098] An agent may be associated with a single copy of a unique label or it may be associated with multiple copies of the same unique label including for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10,000, 100,000 or more copies of the same unique label. In this context, the label is considered unique because it is different from labels associated with other, different agents.
[0099] Attachment of labels to agents may be direct or indirect. The attachment chemistry will depend on the nature of the agent and/or any derivatisation or functionalisation applied to the agent. For example, labels can be directly attached through covalent attachment. The label may include a moiety, which may be a non-nucleotide chemical modification, to facilitate attachment. By way of non-limiting example, the label may include methylated nucleotides, uracil bases, phosphorothioate groups, ribonucleotides, diol linkages, disulphide linkages, etc., to enable covalent attachment to an agent.
[0100] In another example, a label can be attached to an agent via a linker or in another indirect manner. Examples of linkers, include, but are not limited to, carbon-containing chains, polyethylene glycol (PEG), nucleic acids, monosaccharide units, and peptides. The linkers may be cleavable under certain conditions. Cleavable linkers are discussed in greater detail herein.
[0101] Methods for attaching nucleic acids to each other, as for example attaching nucleic acid labels to nucleic acid agents, are known in the art. Such methods include but are not limited to ligation and polymerase-mediated attachment methods (see, e.g., U.S. Patent Nos. 7863058 and 7754429; Green and Sambrook. Molecular Cloning: A Laboratory Manual, Fourth Edition, 2012; Current Protocols in Molecular Biology, and Current Protocols in Nucleic Acid Chemistry, all of which are incorporated herein by reference).
Detectable Oligonucleotide Tags
[0102] The unique labels of the invention are, at least in part, nucleic acid in nature, and are generated by sequentially attaching two or more detectable oligonucleotide tags to each other. As used herein, a detectable oligonucleotide tag is an oligonucleotide that can be detected by sequencing of its nucleotide sequence and/or by detecting non-nucleic acid detectable moieties it may be attached to.
[0103] The oligonucleotides tags are typically randomly selected from a diverse plurality of oligonucleotide tags. In some instances, an oligonucleotide tag may be present once in a plurality or it may be present multiple times in a plurality. In the latter instance, the plurality of tags may be comprised of a number of subsets each comprising a plurality of identical tags. In some important embodiments, these subsets are physically separate from each other. Physical separation may be achieved by providing the subsets in separate droplets from an emulsion. It is the random selection and thus combination of oligonucleotide tags that results in a unique label. Accordingly, the number of distinct (i.e., different) oligonucleotide tags required to uniquely label a plurality of agents can be far less than the number of agents being labeled. This is particularly advantageous when the number of agents is large (e.g., when the agents are members of a library). [0104] As used herein, the term "oligonucleotide" refers to a nucleic acid such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA/RNA hybrids and includes analogs of either DNA or RNA made from nucleotide analogs known in the art (see, e.g. U.S. Patent or Patent Application Publications: US 7399845, US 7741457, US 8022193, US 7569686, US 7335765, US 7314923, US 7335765, and US 7816333, US 20110009471, the entire contents of each of which are incorporated herein by reference). Oligonucleotides may be single-stranded (such as sense or antisense oligonucleotides), double-stranded, or partially single-stranded and partially double-stranded.
[0105] A unique nucleotide sequence may be a nucleotide sequence that is different (and thus distinguishable) from the sequence of each detectable oligonucleotide tag in a plurality of detectable oligonucleotide tags. A unique nucleotide sequence may also be a nucleotide sequence that is different (and thus distinguishable) from the sequence of each detectable oligonucleotide tag in a first plurality of detectable oligonucleotide tags but identical to the sequence of at least one detectable oligonucleotide tag in a second plurality of detectable oligonucleotide tags. A unique sequence may differ from other sequences by multiple bases (or base pairs). The multiple bases may be contiguous or non-contiguous. Methods for obtaining nucleotide sequences (e.g., sequencing methods) are described herein and/or are known in the art.
[0106] In some embodiments, detectable oligonucleotide tags comprise one or more of a ligation sequence, a priming sequence, a capture sequence, and a unique sequence (optionally referred to herein as an index sequence). A ligation sequence is a sequence complementary to a second nucleotide sequence which allows for ligation of the detectable oligonucleotide tag to another entity comprising the second nucleotide sequence, e.g., another detectable oligonucleotide tag or an oligonucleotide adapter. A priming sequence is a sequence complementary to a primer, e.g., an oligonucleotide primer used for an amplification reaction such as but not limited to PCR. A capture sequence is a sequence capable of being bound by a capture entity. A capture entity may be an oligonucleotide comprising a nucleotide sequence complementary to a capture sequence, e.g. a second detectable oligonucleotide tag. A capture entity may also be any other entity capable of binding to the capture sequence, e.g. an antibody or peptide. An index sequence is a sequence comprising a unique nucleotide sequence and/or a detectable moiety as described above. [0107] "Complementary" is a term which is used to indicate a sufficient degree of complementarity between two nucleotide sequences such that stable and specific binding occurs between one and preferably more bases (or nucleotides, as the terms are used interchangeably herein) of the two sequences. For example, if a nucleotide in a first nucleotide sequence is capable of hydrogen bonding with a nucleotide in second nucleotide sequence, then the bases are considered to be complementary to each other. Complete (i.e., 100%) complementarity between a first nucleotide sequence and a second nucleotide is preferable, but not required for ligation, priming, or capture sequences.
[0108] Table 1 below provides examples of certain oligonucleotide tags of the invention:
Table 1
Tag Full Oligo Sequence (Top) Full Oligo Sequence (Bot)
GGATATCTGTGGATATCTGT /5Phos/CAGAGGATATCTGTGGATATCTGT /5Phos/CGAAACAGATATCCACAGATATCC GGCTGCCATAGGCTGCCATA /5Phos/CAGAGGCTGCCATAGGCTGCCATA /5Phos/CGAATATGGCAGCCTATGGCAGCC GGTATGTCAAGGTATGTCAA /5Phos/CAGAGGTATGTCAAGGTATGTCAA /5Phos/CGAATTGACATACCTTGACATACC GGTGAAGGACGGTGAAGGAC /5Phos/CAGAGGTGAAGGACGGTGAAGGAC /5Phos/CGAAGTCCTTCACCGTCCTTCACC GTACCAGCATGTACCAGCAT /5Phos/CAGAGTACCAGCATGTACCAGCAT /5Phos/CGAAATGCTGGTACATGCTGGTAC GTATACATCCGTATACATCC /5Phos/CAGAGTATACATCCGTATACATCC /5Phos/CGAAGGATGTATACGGATGTATAC GTATCGCTCAGTATCGCTCA /5Phos/CAGAGTATCGCTCAGTATCGCTCA /5Phos/CGAATGAGCGATACTGAGCGATAC GTCCGATGGAGTCCGATGGA /5Phos/CAGAGTCCGATGGAGTCCGATGGA /5Phos/CGAATCCATCGGACTCCATCGGAC GTGTGGAATTGTGTGGAATT /5Phos/CAGAGTGTGGAATTGTGTGGAATT /5Phos/CGAAAATTCCACACAATTCCACAC GTGTGGCAGAGTGTGGCAGA /5Phos/CAGAGTGTGGCAGAGTGTGGCAGA /5Phos/CGAATCTGCCACACTCTGCCACAC GTTACGGTGAGTTACGGTGA /5Phos/CAGAGTTACGGTGAGTTACGGTGA /5Phos/CGAATCACCGTAACTCACCGTAAC GTTCGTACATGTTCGTACAT /5Phos/CAGAGTTCGTACATGTTCGTACAT /5Phos/CGAAATGTACGAACATGTACGAAC TAACCGGTAATAACCGGTAA /5Phos/CAGATAACCGGTAATAACCGGTAA /5Phos/CGAATTACCGGTTATTACCGGTTA TACAGAGTCATACAGAGTCA /5Phos/CAGATACAGAGTCATACAGAGTCA /5Phos/CGAATGACTCTGTATGACTCTGTA TACCGAGATATACCGAGATA /5Phos/CAGATACCGAGATATACCGAGATA /5Phos/CGAATATCTCGGTATATCTCGGTA TACGCATCGCTACGCATCGC /5Phos/CAGATACGCATCGCTACGCATCGC /5Phos/CGAAGCGATGCGTAGCGATGCGTA TACGCGCTTGTACGCGCTTG /5Phos/CAGATACGCGCTTGTACGCGCTTG /5Phos/CGAACAAGCGCGTACAAGCGCGTA TAGACTTCACTAGACTTCAC /5Phos/CAGATAGACTTCACTAGACTTCAC /5Phos/CGAAGTGAAGTCTAGTGAAGTCTA TATCGCTTAGTATCGCTTAG /5Phos/CAGATATCGCTTAGTATCGCTTAG /5Phos/CGAACTAAGCGATACTAAGCGATA TCACGATCCGTCACGATCCG /5Phos/CAGATCACGATCCGTCACGATCCG /5Phos/CGAACGGATCGTGACGGATCGTGA TCAGCTTCGCTCAGCTTCGC /5Phos/CAGATCAGCTTCGCTCAGCTTCGC /5Phos/CGAAGCGAAGCTGAGCGAAGCTGA TCCGGACTTCTCCGGACTTC /5Phos/CAGATCCGGACTTCTCCGGACTTC /5Phos/CGAAGAAGTCCGGAGAAGTCCGGA TCCGGTATGGTCCGGTATGG /5Phos/CAGATCCGGTATGGTCCGGTATGG /5Phos/CGAACCATACCGGACCATACCGGA TCCGTGGTGATCCGTGGTGA /5Phos/CAGATCCGTGGTGATCCGTGGTGA /5Phos/CGAATCACCACGGATCACCACGGA TCCTCATCCATCCTCATCCA /5Phos/CAGATCCTCATCCATCCTCATCCA /5Phos/CGAATGGATGAGGATGGATGAGGA TCCTTCTGGATCCTTCTGGA /5Phos/CAGATCCTTCTGGATCCTTCTGGA /5Phos/CGAATCCAGAAGGATCCAGAAGGA TCGCTGTTGCTCGCTGTTGC /5Phos/CAGATCGCTGTTGCTCGCTGTTGC /5Phos/CGAAGCAACAGCGAGCAACAGCGA TCGGAGCTTGTCGGAGCTTG /5Phos/CAGATCGGAGCTTGTCGGAGCTTG /5Phos/CGAACAAGCTCCGACAAGCTCCGA TCGGCATGAGTCGGCATGAG /5Phos/CAGATCGGCATGAGTCGGCATGAG /5Phos/CGAACTCATGCCGACTCATGCCGA TCGGTGGAACTCGGTGGAAC /5PhoS/CAGATCGGTGGAACTCGGTGGAAC /5Phos/CGAAGTTCCACCGAGTTCCACCGA TCGGTTGTAATCGGTTGTAA /5Phos/CAGATCGGTTGTAATCGGTTGTAA /5Phos/CGAATTACAACCGATTACAACCGA TCGTGTGGCATCGTGTGGCA /5Phos/CAGATCGTGTGGCATCGTGTGGCA /5Phos/CGAATGCCACACGATGCCACACGA TCTGTTCCGCTCTGTTCCGC /5Phos/CAGATCTGTTCCGCTCTGTTCCGC /5Phos/CGAAGCGGAACAGAGCGGAACAGA TCTTGGCGTCTCTTGGCGTC /5Phos/CAGATCTTGGCGTCTCTTGGCGTC /5Phos/CGAAGACGCCAAGAGACGCCAAGA TGATTGAGTCTGATTGAGTC /5Phos/CAGATGATTGAGTCTGATTGAGTC /5Phos/CGAAGACTCAATCAGACTCAATCA TGCGTTGGCATGCGTTGGCA /5Phos/CAGATGCGTTGGCATGCGTTGGCA /5Phos/CGAATGCCAACGCATGCCAACGCA TGGACATGCGTGGACATGCG /5Phos/CAGATGGACATGCGTGGACATGCG /5Phos/CGAACGCATGTCCACGCATGTCCA TGGCAAGCCATGGCAAGCCA /5Phos/CAGATGGCAAGCCATGGCAAGCCA /5Phos/CGAATGGCTTGCCATGGCTTGCCA TGGTTAACTGTGGTTAACTG /5Phos/CAGATGGTTAACTGTGGTTAACTG /5Phos/CGAACAGTTAACCACAGTTAACCA TGTACCTGAGTGTACCTGAG /5Phos/CAGATGTACCTGAGTGTACCTGAG /5Phos/CGAACTCAGGTACACTCAGGTACA TGTACTACAGTGTACTACAG /5Phos/CAGATGTACTACAGTGTACTACAG /5Phos/CGAACTGTAGTACACTGTAGTACA TGTCGGTTGCTGTCGGTTGC /5Phos/CAGATGTCGGTTGCTGTCGGT GC /5Phos/CGAAGCAACCGACAGCAACCGACA TGTCTGTCGGTGTCTGTCGG /5Phos/CAGATGTCTGTCGGTGTCTGTCGG /5Phos/CGAACCGACAGACACCGACAGACA TGTGACTATCTGTGACTATC /5Phos/CAGATGTGACTATCTGTGACTATC / 5Phos/CGAAGATAGTCACAGATAGTCACA TGTGAGTGCGTGTGAGTGCG / 5Phos /CAGATGTGAGTGCGTGTGAGTGCG /5Phos/CGAACGCACTCACACGCACTCACA TGTGGTTCGCTGTGGTTCGC /5Phos/CAGATGTGGTTCGCTGTGGTTCGC /5Phos/CGAAGCGAACCACAGCGAACCACA TGTTCTCTACTGTTCTCTAC /5Phos/CAGATGTTCTCTACTGTTCTCTAC /5Phos/CGAAGTAGAGAACAGTAGAGAACA TTAGCCAGTCTTAGCCAGTC / 5Phos/CAGATTAGCCAGTCTTAGCCAGTC /5Phos/CGAAGACTGGCTAAGACTGGCTAA TTCGCGAATATTCGCGAATA /5Phos/CAGATTCGCGAATATTCGCGAATA /5Phos/CGAATATTCGCGAATATTCGCGAA TTCGCGGTACTTCGCGGTAC /5Phos/CAGATTCGCGGTACTTCGCGGTAC /5Phos/CGAAGTACCGCGAAGTACCGCGAA TTCTATGCAGTTCTATGCAG /5Phos/CAGATTCTATGCAGTTCTATGCAG /5Phos/CGAACTGCATAGAACTGCATAGAA TTCTTACCGATTCTTACCGA / 5Phos/CAGATTCTTACCGATTCTTACCGA /5Phos/CGAATCGGTAAGAATCGGTAAGAA TTGCTCTGGATTGCTCTGGA /5Phos/CAGATTGCTCTGGATTGCTCTGGA /5Phos/CGAATCCAGAGCAATCCAGAGCAA TTGTAACACCTTGTAACACC /5Phos /CAGATTGTAACACCTTGTAACACC / 5Phos/CGAAGGTGTTACAAGGTGTTACAA AACAAGTTCCAACAAGTTCC /5PhoS/CAGAAACAAGTTCCAACAAGTTCC / 5Phos /CGAAGGAACTTGTTGGAACTTGTT AACAGCACGCAACAGCACGC /5Phos/CAGAAACAGCACGCAACAGCACGC / 5Phos/CGAAGCGTGCTGTTGCGTGCTGTT AACGTGCGGTAACGTGCGGT /5Phos/CAGAAACGTGCGGTAACGTGCGGT /5Phos/CGAAACCGCACGTTACCGCACGTT AAGAGCGATGAAGAGCGATG /5Phos/CAGAAAGAGCGATGAAGAGCGATG /5Phos/CGAACATCGCTCTTCATCGCTCTT AAGGAATTCCAAGGAATTCC /5Phos /CAGAAAGGAATTCCAAGGAATTCC / 5Phos/CGAAGGAATTCCTTGGAATTCCTT AAGTGAGGCGAAGTGAGGCG /5PhoS/CAGAAAGTGAGGCGAAGTGAGGCG /5Phos/CGAACGCCTCACTTCGCCTCACTT AATACGCGATAATACGCGAT /5Phos/CAGAAATACGCGATAATACGCGAT / 5Phos/CGAAATCGCGTATTATCGCGTATT AATAGGTTGGAATAGGTTGG /5Phos/CAGAAATAGGTTGGAATAGGTTGG /5Phos/CGAACCAACCTATTCCAACCTATT AATCGAAGCTAATCGAAGCT /5Phos/CAGAAATCGAAGCTAATCGAAGCT /5Phos/CGAAAGCTTCGATTAGCTTCGATT ACACTACGATACACTACGAT /5Phos/CAGAACACTACGATACACTACGAT /5Phos/CGAAATCGTAGTGTATCGTAGTGT ACACTCAGCCACACTCAGCC /5Phos/CAGAACACTCAGCCACACTCAGCC /5Phos/CGAAGGCTGAGTGTGGCTGAGTGT ACATCGAGCCACATCGAGCC /5Phos/CAGAACATCGAGCCACATCGAGCC /5Phos/CGAAGGCTCGATGTGGCTCGATGT ACATTATGCGACATTATGCG /5Phos/CAGAACATTATGCGACATTATGCG / 5Phos/CGAACGCATAATGTCGCATAATGT ACCAGCAACTACCAGCAACT /5Phos/CAGAACCAGCAACTACCAGCAACT /5Phos/CGAAAGTTGCTGGTAGTTGCTGGT ACCGAACACGACCGAACACG /5Phos/CAGAACCGAACACGACCGAACACG /5Phos/CGAACGTGTTCGGTCGTGTTCGGT ACCGAACGGTACCGAACGGT /5Phos/CAGAACCGAACGGTACCGAACGGT /5Phos/CGAAACCGTTCGGTACCGTTCGGT ACGAGTGGCTACGAGTGGCT /5Phos/CAGAACGAGTGGCTACGAGTGGCT /5Phos/CGAAAGCCACTCGTAGCCACTCGT ACGGTTACGGACGGTTACGG /5Phos/CAGAACGGTTACGGACGGTTACGG /5Phos/CGAACCGTAACCGTCCGTAACCGT ACGTAAGCGCACGTAAGCGC /5Phos/CAGAACGTAAGCGCACGTAAGCGC / 5Phos/CGAAGCGCTTACGTGCGCTTACGT ACTATGTGCTACTATGTGCT /5Phos/CAGAACTATGTGCTACTATGTGCT / 5Phos/CGAAAGCACATAGTAGCACATAGT ACTGGAGTCCACTGGAGTCC /5Phos/CAGAACTGGAGTCCACTGGAGTCC /5Phos/CGAAGGACTCCAGTGGACTCCAGT ACTGTACTTCACTGTACTTC /5Phos/CAGAACTGTACTTCACTGTACTTC /5Phos/CGAAGAAGTACAGTGAAGTACAGT AGAAGTCTGCAGAAGTCTGC /5Phos/CAGAAGAAGTCTGCAGAAGTCTGC /5Phos /CGAAGCAGACTTCTGCAGACTTCT AGACGCGAGTAGACGCGAGT / 5Phos/CAGAAGACGCGAGTAGACGCGAGT /5Phos/CGAAACTCGCGTCTACTCGCGTCT AGACTGTGCTAGACTGTGCT /5Phos /CAGAAGACTGTGCTAGACTGTGCT /5Phos /CGAAAGCACAGTCTAGCACAGTCT AGAGTACGCCAGAGTACGCC /5Phos/CAGAAGAGTACGCCAGAGTACGCC / 5Phos /CGAAGGCGTACTCTGGCGTACTCT AGATCGACTGAGATCGACTG /5Phos/CAGAAGATCGACTGAGATCGACTG / 5Phos /CGAACAGTCGATCTCAGTCGATCT AGATTCGGCCAGATTCGGCC /5Phos/CAGAAGATTCGGCCAGATTCGGCC /5Phos/CGAAGGCCGAATCTGGCCGAATCT AGCAAGTGCTAGCAAGTGCT /5Phos/CAGAAGCAAGTGCTAGCAAGTGCT /5PhoS /CGAAAGCACTTGCTAGCACTTGCT AGCCATTGCGAGCCATTGCG /5Phos/CAGAAGCCATTGCGAGCCATTGCG /5Phos/CGAACGCAATGGCTCGCAATGGCT AGCGGACAGTAGCGGACAGT / 5Phos/CAGAAGCGGACAGTAGCGGACAGT /5Phos /CGAAACTGTCCGCTACTGTCCGCT AGCGTAAGCGAGCGTAAGCG / 5Phos/CAGAAGCGTAAGCGAGCGTAAGCG /5Phos /CGAACGCTTACGCTCGCTTACGCT AGCTATTCTCAGCTATTCTC /5Phos /CAGAAGCTATTCTCAGCTATTCTC /5Phos/CGAAGAGAATAGCTGAGAATAGCT AGCTTATACGAGCTTATACG / 5Phos /CAGAAGCTTATACGAGCTTATACG /5Phos/CGAACGTATAAGCTCGTATAAGCT AGGACAGATGAGGACAGATG /5Phos /CAGAAGGACAGATGAGGACAGATG /5Phos/CGAACATCTGTCCTCATCTGTCCT AGGATCAGATAGGATCAGAT /5Phos /CAGAAGGATCAGATAGGATCAGAT /5Phos/CGAAATCTGATCCTATCTGATCCT AGGCTGAAGGAGGCTGAAGG / 5Phos /CAGAAGGCTGAAGGAGGCTGAAGG /5Phos/CGAACCTTCAGCCTCCTTCAGCCT AGGTACGAGGAGGTACGAGG /5Phos/CAGAAGGTACGAGGAGGTACGAGG /5Phos/CGAACCTCGTACCTCCTCGTACCT AGGTAGGCTCAGGTAGGCTC /5Phos/CAGAAGGTAGGCTCAGGTAGGCTC /5Phos/CGAAGAGCCTACCTGAGCCTACCT AGGTGAACGGAGGTGAACGG /5Phos /CAGAAGGTGAACGGAGGTGAACGG /5Phos/CGAACCGTTCACCTCCGTTCACCT AGGTGCCAATAGGTGCCAAT / 5Phos/CAGAAGGTGCCAATAGGTGCCAAT /5Phos/CGAAATTGGCACCTATTGGCACCT AGGTTCAACGAGGTTCAACG / 5Phos/CAGAAGGTTCAACGAGGTTCAACG /5Phos /CGAACGTTGAACCTCGTTGAACCT AGTCACTCGCAGTCACTCGC /5Phos/CAGAAGTCACTCGCAGTCACTCGC /5Phos/CGAAGCGAGTGACTGCGAGTGACT AGTCGTGGTGAGTCGTGGTG /5Phos/CAGAAGTCGTGGTGAGTCGTGGTG /5Phos/CGAACACCACGACTCACCACGACT AGTGGAGGAGAGTGGAGGAG /5Phos/CAGAAGTGGAGGAGAGTGGAGGAG /5Phos/CGAACTCCTCCACTCTCCTCCACT AGTTGAGCGCAGTTGAGCGC /5Phos/CAGAAGTTGAGCGCAGTTGAGCGC /5Phos/CGAAGCGCTCAACTGCGCTCAACT AGTTGATGGTAGTTGATGGT /5Phos/CAGAAGTTGATGGTAGTTGATGGT /5Phos /CGAAACCATCAACTACCATCAACT ATAACACAGCATAACACAGC /5Phos/CAGAATAACACAGCATAACACAGC /5PhoS /CGAAGCTGTGTTATGCTGTGTTAT ATAAGGTCCGATAAGGTCCG /5Phos/CAGAATAAGGTCCGATAAGGTCCG /5PhoΞ/CGAACGGACCTTATCGGACCTTAT ATACAGTCAGATACAGTCAG /5Phos/CAGAATACAGTCAGATACAGTCAG /5Phos/CGAACTGACTGTATCTGACTGTAT ATACCGGCCTATACCGGCCT /5Phos/CAGAATACCGGCCTATACCGGCCT /5Phos/CGAAAGGCCGGTATAGGCCGGTAT ATAGCAGGATATAGCAGGAT /5Phos/CAGAATAGCAGGATATAGCAGGAT /5Phos/CGAAATCCTGCTATATCCTGCTAT ATAGCGTTACATAGCGTTAC /5Phos/CAGAATAGCGTTACATAGCGTTAC /5Phos/CGAAGTAACGCTATGTAACGCTAT ATAGTCCAACATAGTCCAAC /5Phos/CAGAATAGTCCAACATAGTCCAAC /5Phos/CGAAGTTGGACTATGTTGGACTAT ATATCGGCGGATATCGGCGG /5Phos/CAGAATATCGGCGGATATCGGCGG /5Phos/CGAACCGCCGATATCCGCCGATAT ATCCGTGATGATCCGTGATG /5Phos/CAGAATCCGTGATGATCCGTGATG /5Phos/CGAACATCACGGATCATCACGGAT ATCGTACCGGATCGTACCGG /5Phos/CAGAATCGTACCGGATCGTACCGG /5Phos/CGAACCGGTACGATCCGGTACGAT ATCGTTACTGATCGTTACTG /5Phos/CAGAATCGTTACTGATCGTTACTG /5PhoS/CGAACAGTAACGATCAGTAACGAT ATGAAGATGCATGAAGATGC /5Phos/CAGAATGAAGATGCATGAAGATGC /5PhoS/CGAAGCATCTTCATGCATCTTCAT ATGGCGTGGTATGGCGTGGT /5Phos/CAGAATGGCGTGGTATGGCGTGGT /5Phos/CGAAACCACGCCATACCACGCCAT ATGTTGAGCGATGTTGAGCG /5Phos/CAGAATGTTGAGCGATGTTGAGCG /5Phos/CGAACGCTCAACATCGCTCAACAT ATTAGCTGTCATTAGCTGTC /5Phos/CAGAATTAGCTGTCATTAGCTGTC /5Phos/CGAAGACAGCTAATGACAGCTAAT ATTCGCGTGCATTCGCGTGC /5Phos/CAGAATTCGCGTGCATTCGCGTGC /5Phos/CGAAGCACGCGAATGCACGCGAAT ATTGCGCTCCATTGCGCTCC /5Phos/CAGAATTGCGCTCCATTGCGCTCC /5Phos/CGAAGGAGCGCAATGGAGCGCAAT ATTGTGTTGCATTGTGTTGC /5Phos/CAGAATTGTGTTGCATTGTGTTGC /5Phos/CGAAGCAACACAATGCAACACAAT CAAGACGAATCAAGACGAAT /5Phos/CAGACAAGACGAATCAAGACGAAT /5Phos/CGAAATTCGTCTTGATTCGTCTTG CACGCACTGTCACGCACTGT /5Phos/CAGACACGCACTGTCACGCACTGT /5Phos/CGAAACAGTGCGTGACAGTGCGTG CACGGAGTAGCACGGAGTAG /5Phos/CAGACACGGAGTAGCACGGAGTAG /5Phos/CGAACTACTCCGTGCTACTCCGTG CACGGTATCGCACGGTATCG /5Phos/CAGACACGGTATCGCACGGTATCG /5Phos/CGAACGATACCGTGCGATACCGTG CACTCGGATTCACTCGGATT /5Phos/CAGACACTCGGATTCACTCGGATT /5Phos/CGAAAATCCGAGTGAATCCGAGTG CACTTGAGTACACTTGAGTA /5Phos/CAGACACTTGAGTACACTTGAGTA /5Phos/CGAATACTCAAGTGTACTCAAGTG CAGCCTCAGTCAGCCTCAGT /5Phos/CAGACAGCCTCAGTCAGCCTCAGT /5Phos/CGAAACTGAGGCTGACTGAGGCTG CAGCTCCAAGCAGCTCCAAG /5Phos/CAGACAGCTCCAAGCAGCTCCAAG /5Phos/CGAACTTGGAGCTGCTTGGAGCTG CATCGAGGAGCATCGAGGAG /5Phos/CAGACATCGAGGAGCATCGAGGAG /5Phos/CGAACTCCTCGATGCTCCTCGATG CATTGGCCGTCATTGGCCGT /5Phos/CAGACATTGGCCGTCATTGGCCGT /5Phos/CGAAACGGCCAATGACGGCCAATG CCAGAACTGGCCAGAACTGG /5Phos/CAGACCAGAACTGGCCAGAACTGG /5Phos/CGAACCAGTTCTGGCCAGTTCTGG CCAGATACGGCCAGATACGG /5Phos/CAGACCAGATACGGCCAGATACGG /5Phos/CGAACCGTATCTGGCCGTATCTGG CCAGGATCCACCAGGATCCA /5Phos/CAGACCAGGATCCACCAGGATCCA /5Phos/CGAATGGATCCTGGTGGATCCTGG CCAGGATGTTCCAGGATGTT /5Phos/CAGACCAGGATGTTCCAGGATGTT /5Phos/CGAAAACATCCTGGAACATCCTGG CCGAGCATGTCCGAGCATGT /5Phos/CAGACCGAGCATGTCCGAGCATGT /5Phos/CGAAACATGCTCGGACATGCTCGG CCGGAGTGTTCCGGAGTGTT /5Phos/CAGACCGGAGTGTTCCGGAGTGTT /5Phos/CGAAAACACTCCGGAACACTCCGG CCGGTACCATCCGGTACCAT /5Phos/CAGACCGGTACCATCCGGTACCAT /5Phos/CGAAATGGTACCGGATGGTACCGG CCGTCTAAGGCCGTCTAAGG /5Phos/CAGACCGTCTAAGGCCGTCTAAGG /5Phos/CGAACCTTAGACGGCCTTAGACGG CCTCCATTAACCTCCATTAA /5Phos/CAGACCTCCATTAACCTCCATTAA /5Phos/CGAATTAATGGAGGTTAATGGAGG CCTCCTGACTCCTCCTGACT /5Phos/CAGACCTCCTGACTCCTCCTGACT /5Phos/CGAAAGTCAGGAGGAGTCAGGAGG CCTCTGCTCTCCTCTGCTCT /5Phos/CAGACCTCTGCTCTCCTCTGCTCT /5Phos/CGAAAGAGCAGAGGAGAGCAGAGG CCTGCCTTGTCCTGCCTTGT /5Phos/CAGACCTGCCTTGTCCTGCCTTGT /5Phos/CGAAACAAGGCAGGACAAGGCAGG CCTGGCCATTCCTGGCCATT /5Phos/CAGACCTGGCCATTCCTGGCCATT /5Phos/CGAAAATGGCCAGGAATGGCCAGG CCTTAACGCGCCTTAACGCG /5Phos/CAGACCTTAACGCGCCTTAACGCG /5Phos/CGAACGCGTTAAGGCGCGTTAAGG CGACCTGTCTCGACCTGTCT /5Phos/CAGACGACCTGTCTCGACCTGTCT /5Phos/CGAAAGACAGGTCGAGACAGGTCG CGCGTTACGTCGCGTTACGT /5Phos/CAGACGCGTTACGTCGCGTTACGT /5Phos/CGAAACGTAACGCGACGTAACGCG CGGCAGTTCACGGCAGTTCA /5Phos/CAGACGGCAGTTCACGGCAGTTCA /5Phos/CGAATGAACTGCCGTGAACTGCCG CGGCCATTAGCGGCCATTAG /5Phos/CAGACGGCCATTAGCGGCCATTAG /5Phos/CGAACTAATGGCCGCTAATGGCCG CGTATTGCATCGTATTGCAT /5Phos/CAGACGTATTGCATCGTATTGCAT /5Phos/CGAAATGCAATACGATGCAATACG CTACGACCGTCTACGACCGT /5Phos/CAGACTACGACCGTCTACGACCGT /5Phos/CGAAACGGTCGTAGACGGTCGTAG CTAGGAAGGTCTAGGAAGGT /5Phos/CAGACTAGGAAGGTCTAGGAAGGT /5Phos/CGAAACCTTCCTAGACCTTCCTAG CTAGTCCTGTCTAGTCCTGT /5Phos/CAGACTAGTCCTGTCTAGTCCTGT /5Phos/CGAAACAGGACTAGACAGGACTAG CTAGTGGAGGCTAGTGGAGG /5Phos/CAGACTAGTGGAGGCTAGTGGAGG /5Phos/CGAACCTCCACTAGCCTCCACTAG CTCGCAGAGTCTCGCAGAGT /5Phos/CAGACTCGCAGAGTCTCGCAGAGT /5Phos/CGAAACTCTGCGAGACTCTGCGAG CTCGCTTCGTCTCGCTTCGT /5Phos/CAGACTCGCTTCGTCTCGCTTCGT /5Phos/CGAAACGAAGCGAGACGAAGCGAG CTCGTTAGCGCTCGTTAGCG /5Phos/CAGACTCGTTAGCGCTCGTTAGCG /5Phos/CGAACGCTAACGAGCGCTAACGAG CTCTTCCAAGCTCTTCCAAG /5Phos/CAGACTCTTCCAAGCTCTTCCAAG /5Phos/CGAACTTGGAAGAGCTTGGAAGAG CTGCTTCAATCTGCTTCAAT /5Phos/CAGACTGCTTCAATCTGCTTCAAT /5PhoS/CGAAATTGAAGCAGATTGAAGCAG CTGGTATCAACTGGTATCAA /5Phos/CAGACTGGTATCAACTGGTATCAA /5Phos/CGAATTGATACCAGTTGATACCAG CTGTCTTCGGCTGTCTTCGG /5Phos/CAGACTGTCTTCGGCTGTCTTCGG /5Phos/CGAACCGAAGACAGCCGAAGACAG CTTCATGACGCTTCATGACG /5Phos/CAGACTTCATGACGCTTCATGACG /5Phos/CGAACGTCATGAAGCGTCATGAAG CTTCGGCAGTCTTCGGCAGT /5Phos/CAGACTTCGGCAGTCTTCGGCAGT /5Phos/CGAAACTGCCGAAGACTGCCGAAG CTTGACCGGTCTTGACCGGT /5Phos/CAGACTTGACCGGTCTTGACCGGT /5Phos/CGAAACCGGTCAAGACCGGTCAAG CTTGCCTATTCTTGCCTATT /5Phos/CAGACTTGCCTATTCTTGCCTATT /5Phos/CGAAAATAGGCAAGAATAGGCAAG GAACTTGTGAGAACTTGTGA /5Phos/CAGAGAACTTGTGAGAACTTGTGA /5Phos/CGAATCACAAGTTCTCACAAGTTC GAAGCATTCTGAAGCATTCT /5Phos/CAGAGAAGCATTCTGAAGCATTCT /5Phos/CGAAAGAATGCTTCAGAATGCTTC GAATCCATTCGAATCCATTC /5Phos/CAGAGAATCCATTCGAATCCATTC /5Phos/CGAAGAATGGATTCGAATGGATTC GACGCCTGTTGACGCCTGTT /5Phos/CAGAGACGCCTGTTGACGCCTGTT /5Phos/CGAAAACAGGCGTCAACAGGCGTC GACGTAGGACGACGTAGGAC /5Phos/CAGAGACGTAGGACGACGTAGGAC /5Phos/CGAAGTCCTACGTCGTCCTACGTC GACTAATGGTGACTAATGGT /5Phos/CAGAGACTAATGGTGACTAATGGT /5Phos/CGAAACCATTAGTCACCATTAGTC GAGCCTCCTTGAGCCTCCTT /5Phos/CAGAGAGCCTCCTTGAGCCTCCTT /5Phos/CGAAAAGGAGGCTCAAGGAGGCTC GAGCGTCTACGAGCGTCTAC /5Phos/CAGAGAGCGTCTACGAGCGTCTAC /5Phos/CGAAGTAGACGCTCGTAGACGCTC GAGGATAGGCGAGGATAGGC /5Phos/CAGAGAGGATAGGCGAGGATAGGC /5Phos/CGAAGCCTATCCTCGCCTATCCTC GAGTGCCATCGAGTGCCATC /5Phos/CAGAGAGTGCCATCGAGTGCCATC /5Phos/CGAAGATGGCACTCGATGGCACTC GAGTGGATCTGAGTGGATCT /5Phos/CAGAGAGTGGATCTGAGTGGATCT /5Phos/CGAAAGATCCACTCAGATCCACTC GAGTGGTAGCGAGTGGTAGC /5Phos/CAGAGAGTGGTAGCGAGTGGTAGC /5Phos/CGAAGCTACCACTCGCTACCACTC GAGTTAGAGAGAGTTAGAGA /5Phos/CAGAGAGTTAGAGAGAGTTAGAGA /5Phos/CGAATCTCTAACTCTCTCTAACTC GCACATCTGCGCACATCTGC /5Phos/CAGAGCACATCTGCGCACATCTGC /5Phos/CGAAGCAGATGTGCGCAGATGTGC GCACCATTACGCACCATTAC /5Phos/CAGAGCACCATTACGCACCATTAC /5Phos/CGAAGTAATGGTGCGTAATGGTGC GCAGCCTATTGCAGCCTATT /5Phos/CAGAGCAGCCTATTGCAGCCTATT /5Phos/CGAAAATAGGCTGCAATAGGCTGC GCAGTATCAAGCAGTATCAA /5Phos/CAGAGCAGTATCAAGCAGTATCAA /5Phos/CGAATTGATACTGCTTGATACTGC GCCGTCGTTAGCCGTCGTTA /5Phos/CAGAGCCGTCGTTAGCCGTCGTTA /5Phos/CGAATAACGACGGCTAACGACGGC GCCTGAGCTAGCCTGAGCTA /5Phos/CAGAGCCTGAGCTAGCCTGAGCTA /5Phos/CGAATAGCTCAGGCTAGCTCAGGC GCGCAAGCAAGCGCAAGCAA /5Phos/CAGAGCGCAAGCAAGCGCAAGCAA /5Phos/CGAATTGCTTGCGCTTGCTTGCGC GCGTTACGACGCGTTACGAC /5Phos/CAGAGCGTTACGACGCGTTACGAC /5Phos/CGAAGTCGTAACGCGTCGTAACGC GCGTTGGATCGCGTTGGATC /5Phos/CAGAGCGTTGGATCGCGTTGGATC /5Phos/CGAAGATCCAACGCGATCCAACGC GCTAGTCGCAGCTAGTCGCA /5Phos/CAGAGCTAGTCGCAGCTAGTCGCA /5Phos/CGAATGCGACTAGCTGCGACTAGC GCTCACTACCGCTCACTACC /5Phos/CAGAGCTCACTACCGCTCACTACC /5Phos/CGAAGGTAGTGAGCGGTAGTGAGC GCTCGAATTAGCTCGAATTA /5Phos/CAGAGCTCGAATTAGCTCGAATTA /5Phos/CGAATAATTCGAGCTAATTCGAGC GCTCGATTCCGCTCGATTCC /5Phos/CAGAGCTCGATTCCGCTCGATTCC /5Phos/CGAAGGAATCGAGCGGAATCGAGC GCTGAGGATCGCTGAGGATC /5Phos/CAGAGCTGAGGATCGCTGAGGATC /5Phos/CGAAGATCCTCAGCGATCCTCAGC GCTTCATTCTGCTTCATTCT /5Phos/CAGAGCTTCATTCTGCTTCATTCT /5Phos/CGAAAGAATGAAGCAGAATGAAGC GCTTGCTATTGCTTGCTATT /5Phos/CAGAGCTTGCTATTGCTTGCTATT /5Phos/CGAAAATAGCAAGCAATAGCAAGC
GCTTGGTTGCGCTTGGTTGC /5Phos/TTCGGCTTGGTTGCGCTTGGTTGC /5Phos/GTCAGCAACCAAGCGCAACCAAGC GGAGTGGTTCGGAGTGGTTC /5Phos/ TCGGGAGTGGTTCGGAGTGGTTC /5Phos/GTCAGAACCACTCCGAACCACTCC GGATAATACCGGATAATACC /5Phos/TTCGGGATAATACCGGATAATACC /5Phos/GTCAGGTATTATCCGGTATTATCC GGATCGTGGTGGATCGTGGT /5Phos/TTCGGGATCGTGGTGGATCGTGGT /5Phos/GTCAACCACGATCCACCACGATCC GGATGATTGTGGATGATTGT /5Phos/ TCGGGATGATTGTGGATGATTGT /5Phos/GTCAACAATCATCCACAATCATCC GGATGCGTTCGGATGCGTTC /5Phos/TTCGGGATGCGTTCGGATGCGTTC /5Phos/GTCAGAACGCATCCGAACGCATCC GGCGAATGTCGGCGAATGTC /5Phos/TTCGGGCGAATGTCGGCGAATGTC /5Phos/GTCAGACATTCGCCGACATTCGCC GGCGATTGGTGGCGATTGGT /5Phos/ TCGGGCGATTGGTGGCGATTGGT /5Phos/GTCAACCAATCGCCACCAATCGCC GGCGGTATCAGGCGGTATCA /5Phos/ TCGGGCGGTATCAGGCGGTATCA /5Phos/GTCATGATACCGCCTGATACCGCC GGCTATCCACGGCTATCCAC /5Phos/ TCGGGCTATCCACGGCTATCCAC /5Phos/GTCAGTGGATAGCCGTGGATAGCC GGCTATTACAGGCTATTACA /5Phos/ TCGGGCTATTACAGGCTATTACA /5Phos/GTCATGTAATAGCCTGTAATAGCC GGCTGAACTCGGCTGAACTC /5Phos/TTCGGGCTGAACTCGGCTGAACTC /5Phos/GTCAGAGTTCAGCCGAGTTCAGCC GGTACAGTCAGGTACAGTCA /5Phos/ TCGGGTACAGTCAGGTACAGTCA /5Phos/GTCATGACTGTACCTGACTGTACC GGTCGAACCTGGTCGAACCT /5Phos/ TCGGGTCGAACCTGGTCGAACCT /5Phos/GTCAAGGTTCGACCAGGTTCGACC GGTCTCTCGTGGTCTCTCGT /5Phos/TTCGGGTCTCTCGTGGTCTCTCGT /5Phos/GTCAACGAGAGACCACGAGAGACC GGTGCTTGTCGGTGCTTGTC /5Phos/TTCGGGTGCTTGTCGGTGCTTGTC /5Phos/GTCAGACAAGCACCGACAAGCACC GGTTCCACTTGGTTCCACTT /5Phos/ TCGGGTTCCACTTGGTTCCACTT /5Phos/GTCAAAGTGGAACCAAGTGGAACC GGTTGCATCCGGTTGCATCC /5Phos/ TCGGGTTGCATCCGGTTGCATCC /5Phos/GTCAGGATGCAACCGGATGCAACC GGTTGGACGTGGTTGGACGT /5Phos/TTCGGGTTGGACGTGGTTGGACGT /5Phos/GTCAACGTCCAACCACGTCCAACC GTAAGAGCAAGTAAGAGCAA /5Phos/ TCGGTAAGAGCAAGTAAGAGCAA /5Phos/GTCATTGCTCTTACTTGCTCTTAC GTAAGGCTTAGTAAGGCTTA /5Phos/ TCGGTAAGGCTTAGTAAGGCTTA /5Phos/GTCATAAGCCTTACTAAGCCTTAC GTAGTCCTCAGTAGTCCTCA /5Phos/ TCGGTAGTCCTCAGTAGTCCTCA /5Phos/GTCATGAGGACTACTGAGGACTAC GTCCGGTTCTGTCCGGTTCT /5Phos/TTCGGTCCGGTTCTGTCCGGTTCT /5Phos/GTCAAGAACCGGACAGAACCGGAC GTCCTACAGCGTCCTACAGC /5Phos/TTCGGTCCTACAGCGTCCTACAGC /5Phos/GTCAGCTGTAGGACGCTGTAGGAC GTCGGATTAAGTCGGATTAA /5Phos/ TCGGTCGGATTAAGTCGGATTAA /5Phos/GTCATTAATCCGACTTAATCCGAC GTGAACAGAAGTGAACAGAA /5Phos/ TCGGTGAACAGAAGTGAACAGAA /5Phos/GTCATTCTGTTCACTTCTGTTCAC GTGCCTACACGTGCCTACAC /5Phos/ TCGGTGCCTACACGTGCCTACAC /5Phos/GTCAGTGTAGGCACGTGTAGGCAC GTGTATCGGAGTGTATCGGA /5Phos/ TCGGTGTATCGGAGTGTATCGGA /5Phos/GTCATCCGATACACTCCGATACAC GTGTCCATGAGTGTCCATGA /5Phos/ TCGGTGTCCATGAGTGTCCATGA /5Phos/GTCATCATGGACACTCATGGACAC GTGTCTAATCGTGTCTAATC /5Phos/ TCGGTGTCTAATCGTGTCTAATC /5Phos/GTCAGATTAGACACGATTAGACAC GTGTTCCTGCGTGTTCCTGC /5Phos/TTCGGTGTTCCTGCGTGTTCCTGC /5Phos/GTCAGCAGGAACACGCAGGAACAC GTGTTCTGCTGTGTTCTGCT /5Phos/TTCGG GTTCTGCTGTGTTCTGCT /5Phos/GTCAAGCAGAACACAGCAGAACAC GTTAAGAGGAGTTAAGAGGA /5Phos/ TCGGTTAAGAGGAGTTAAGAGGA /5Phos/GTCATCCTCTTAACTCCTCTTAAC GTTAATGCGTGTTAATGCGT /5Phos/TTCGGTTAATGCGTGTTAATGCGT /5Phos/GTCAACGCATTAACACGCATTAAC GTTAGGCTGTGTTAGGCTGT /5Phos/ TCGGTTAGGCTGTGTTAGGCTGT /5Phos/GTCAACAGCCTAACACAGCCTAAC GTTCATTGGAGTTCATTGGA /5Phos/ TCGGTTCATTGGAGTTCATTGGA /5Phos/GTCATCCAATGAACTCCAATGAAC GTTCGGACCAGTTCGGACCA /5Phos/ TCGGTTCGGACCAGTTCGGACCA /5Phos/GTCATGGTCCGAACTGGTCCGAAC GTTGGCCAGTGTTGGCCAGT /5Phos/ TCGGTTGGCCAGTGTTGGCCAGT /5PhoS/GTCAACTGGCCAACACTGGCCAAC GTTGGTAGTTGTTGGTAGTT /5Phos/TTCGGTTGGTAGTTGTTGGTAGTT /5Phos/GTCAAACTACCAACAACTACCAAC TAACACGACATAACACGACA /5Phos/ TCGTAACACGACATAACACGACA /5Phos/GTCATGTCGTGTTATGTCGTGTTA TAAGAGAGCATAAGAGAGCA /5Phos/ TCGTAAGAGAGCATAAGAGAGCA /5Phos/GTCATGCTCTCTTATGCTCTCTTA TAAGAGGCGGTAAGAGGCGG /5Phos/ TCGTAAGAGGCGGTAAGAGGCGG /5Phos/GTCACCGCCTCTTACCGCCTCTTA TAAGGAATGGTAAGGAATGG /5Phos/TTCGTAAGGAATGGTAAGGAATGG /5Phos/GTCACCATTCCTTACCATTCCTTA TAATGAGCACTAATGAGCAC /5Phos/ TCGTAATGAGCACTAATGAGCAC /5Phos/GTCAGTGCTCATTAGTGCTCATTA TACACTGGTCTACACTGGTC /5Phos/TTCGTACACTGGTCTACACTGGTC /5Phos/GTCAGACCAGTGTAGACCAGTGTA TACAGCGCAATACAGCGCAA /5Phos/ TCGTACAGCGCAATACAGCGCAA /5Phos/GTCATTGCGCTGTATTGCGCTGTA TACAGGTTAGTACAGGTTAG /5Phos/ TCGTACAGGTTAGTACAGGTTAG /5Phos/GTCACTAACCTGTACTAACCTGTA TACATTACCGTACATTACCG /5Phos/ TCGTACATTACCGTACATTACCG /5Phos/GTCACGGTAATGTACGGTAATGTA TACCAATCTCTACCAATCTC /5Phos/TTCGTACCAATCTCTACCAATCTC /5Phos/GTCAGAGATTGGTAGAGATTGGTA TACCGGAGAGTACCGGAGAG /5Phos/ TCGTACCGGAGAGTACCGGAGAG /5Phos/GTCACTCTCCGGTACTCTCCGGTA TACCGGCTTCTACCGGCTTC /5Phos/TTCGTACCGGCTTCTACCGGCTTC /5Phos/GTCAGAAGCCGGTAGAAGCCGGTA TACCTACATGTACCTACATG /5Phos/TTCGTACCTACATGTACCTACATG /5Phos/GTCACATGTAGGTACATGTAGGTA TACGATTACGTACGATTACG /5Phos/ TCGTACGATTACGTACGATTACG /5Phos/GTCACGTAATCGTACGTAATCGTA TACGCAGAGGTACGCAGAGG /5Phos/ TCGTACGCAGAGGTACGCAGAGG /5Phos/GTCACCTCTGCGTACCTCTGCGTA TACTCAACGATACTCAACGA /5PhoS/ TCGTACTCAACGATACTCAACGA /5Phos/GTCATCGTTGAGTATCGTTGAGTA TAGACCAACCTAGACCAACC /5Phos/TTCGTAGACCAACCTAGACCAACC /5Phos/GTCAGGTTGGTCTAGGTTGGTCTA TAGACCTCCGTAGACCTCCG /5Phos/TTCGTAGACCTCCGTAGACCTCCG /5Phos/GTCACGGAGGTCTACGGAGGTCTA TAGCAGTTGATAGCAGTTGA /5Phos/ TCGTAGCAGTTGATAGCAGTTGA /5Phos/GTCATCAACTGCTATCAACTGCTA TAGCGCAAGCTAGCGCAAGC /5Phos/TTCGTAGCGCAAGCTAGCGCAAGC /5Phos/GTCAGCTTGCGCTAGCTTGCGCTA TAGCTATACCTAGCTATACC /5Phos/TTCGTAGCTATACCTAGCTATACC /5Phos/GTCAGGTATAGCTAGGTATAGCTA TAGCTTGCGCTAGCTTGCGC /5Phos/ TCGTAGCTTGCGCTAGCTTGCGC /5Phos/GTCAGCGCAAGCTAGCGCAAGCTA TAGGAATCCATAGGAATCCA /5Phos/ TCGTAGGAATCCATAGGAATCCA /5Phos/GTCATGGATTCCTATGGATTCCTA TAGGCCGTTCTAGGCCGTTC /5Phos/TTCGTAGGCCGTTCTAGGCCGTTC /5Phos/GTCAGAACGGCCTAGAACGGCCTA TAGGTCACTATAGGTCACTA /5Phos/TTCGTAGGTCACTATAGGTCACTA /5Phos/GTCATAGTGACCTATAGTGACCTA TAGTCGCGTCTAGTCGCGTC /5Phos/ TCGTAGTCGCGTCTAGTCGCGTC /5Phos/GTCAGACGCGACTAGACGCGACTA TATACGGCTATATACGGCTA /5Phos/TTCGTATACGGCTATATACGGCTA /5Phos/GTCATAGCCGTATATAGCCGTATA TATCGCGGCATATCGCGGCA /5Phos/ TCGTATCGCGGCATATCGCGGCA /5Phos/GTCATGCCGCGATATGCCGCGATA TATGGAGCAATATGGAGCAA /5Phos/ TCGTATGGAGCAATATGGAGCAA /5Phos/GTCATTGCTCCATATTGCTCCATA TATGGCGTGGTATGGCGTGG /5Phos/ TCGTATGGCGTGGTATGGCGTGG /5Phos/GTCACCACGCCATACCACGCCATA TATGTTCAGGTATGTTCAGG /5Phos/ TCGTATGTTCAGGTATGTTCAGG /5Phos/GTCACCTGAACATACCTGAACATA TATTCCTGTCTATTCCTGTC /5Phos/TTCGTATTCCTGTCTATTCCTGTC /5Phos/GTCAGACAGGAATAGACAGGAATA TCAAGAGATCTCAAGAGATC /5Phos/ TCGTCAAGAGATCTCAAGAGATC /5Phos/GTCAGATCTCTTGAGATCTCTTGA TCACTACCAATCACTACCAA /5Phos/ TCGTCACTACCAATCACTACCAA /5Phos/GTCATTGGTAGTGATTGGTAGTGA TCAGTCTGCGTCAGTCTGCG /5Phos/ TCGTCAGTCTGCGTCAGTCTGCG /5Phos/GTCACGCAGACTGACGCAGACTGA TCAGTTAAGCTCAGTTAAGC /5Phos/ TCGTCAGTTAAGCTCAGTTAAGC /5Phos/GTCAGCTTAACTGAGCTTAACTGA TCCAGAGTGGTCCAGAGTGG /5Phos/ TCGTCCAGAGTGGTCCAGAGTGG /5Phos/GTCACCACTCTGGACCACTCTGGA TCCAGTCGTCTCCAGTCGTC /5Phos/TTCGTCCAGTCGTCTCCAGTCGTC /5Phos/GTCAGACGACTGGAGACGACTGGA TCCGACGTTGTCCGACGTTG /5Phos/TTCGTCCGACGTTGTCCGACGTTG /5Phos/GTCACAACGTCGGACAACGTCGGA TCCTACCGACTCCTACCGAC /5Phos/TTCGTCCTACCGACTCCTACCGAC /5Phos/GTCAGTCGGTAGGAGTCGGTAGGA TCCTTCCTCCTCCTTCCTCC /5Phos/TTCGTCCTTCCTCCTCCTTCCTCC /5Phos/GTCAGGAGGAAGGAGGAGGAAGGA TCGAGAGCCATCGAGAGCCA /5Phos/TTCGTCGAGAGCCATCGAGAGCCA /5Phos/GTCATGGCTCTCGATGGCTCTCGA TCGCACAGACTCGCACAGAC /5Phos/TTCGTCGCACAGACTCGCACAGAC /5Phos/GTCAGTCTGTGCGAGTCTGTGCGA TCGCCGTTAGTCGCCGTTAG /5Phos/TTCGTCGCCGTTAGTCGCCGTTAG /5Phos/GTCACTAACGGCGACTAACGGCGA TCGGCACAACTCGGCACAAC /5Phos/ TCGTCGGCACAACTCGGCACAAC /5Phos/GTCAGTTGTGCCGAGTTGTGCCGA TCGGCAGTTGTCGGCAGTTG /5Phos/TTCGTCGGCAGTTGTCGGCAGTTG /5Phos/GTCACAACTGCCGACAACTGCCGA TCGGTCACACTCGGTCACAC /5Phos/ TCGTCGGTCACACTCGGTCACAC /5Phos/GTCAGTGTGACCGAGTGTGACCGA TCGTGCTAGCTCGTGCTAGC /5Phos/ TCGTCGTGCTAGCTCGTGCTAGC /5Phos/GTCAGCTAGCACGAGCTAGCACGA TCTAGCCTAATCTAGCCTAA /5Phos/TTCGTCTAGCCTAATCTAGCCTAA /5Phos/GTCATTAGGCTAGATTAGGCTAGA TCTCACTGCGTCTCACTGCG /5Phos/ TCGTCTCACTGCGTCTCACTGCG /5Phos/GTCACGCAGTGAGACGCAGTGAGA TCTCCGGCAATCTCCGGCAA /5Phos/ TCGTCTCCGGCAATCTCCGGCAA /5Phos/GTCATTGCCGGAGATTGCCGGAGA TCTCGCTCTCTCTCGCTCTC /5Phos/ TCGTCTCGCTCTCTCTCGCTCTC /5Phos/GTCAGAGAGCGAGAGAGAGCGAGA TCTGTCGCAATCTGTCGCAA /5Phos/ TCGTCTGTCGCAATCTGTCGCAA /5Phos/GTCATTGCGACAGATTGCGACAGA TCTTAACCTCTCTTAACCTC /5Phos/TTCGTCTTAACCTCTCTTAACCTC /5Phos/GTCAGAGGTTAAGAGAGGTTAAGA TGACACATGATGACACATGA /5Phos/ TCGTGACACATGATGACACATGA /5Phos/GTCATCATGTGTCATCATGTGTCA TGAGAGTGAATGAGAGTGAA /5Phos/TTCGTGAGAGTGAATGAGAGTGAA /5Phos/GTCATTCACTCTCATTCACTCTCA TGAGTGGCTGTGAGTGGCTG / 5 Pho S / TCGTGAGTGGCTGTGAGTGGCTG / 5 Phos /GTCACAGCCACTCACAGCCACTCA TGATTGAC C ATGATTGAC C A / 5 Pho s / TCGTGATTGACCATGATTGACCA / 5 Phos / GTCATGGTCAATCATGGTCAATCA TGCAGTCGCATGCAGTCGCA / 5 Phos / TCGTGCAGTCGCATGCAGTCGCA / 5 Phos / GTCATGCGACTGCATGCGACTGCA TGCCATGAGGTGCCATGAGG / 5 Phos / TTCGTGCCATGAGGTGCCATGAGG / 5 Phos / GTCACCTCATGGCACCTCATGGCA TGCCTAACGCTGCCTAACGC / 5 Phos /TTCGTGCCTAACGCTGCCTAACGC / 5 Phos / GTCAGCGTTAGGCAGCGTTAGGCA TGCCTATAACTGCCTATAAC / 5 Phos /TTCGTGCCTATAACTGCCTATAAC / 5 Phos / GTCAGTTATAGGCAGTTATAGGCA TGCGAGAGAGTGCGAGAGAG / 5 Phos / TCGTGCGAGAGAGTGCGAGAGAG / 5 Pho s / GTCACTCTCTCGCACTCTCTCGCA TGCGCGATCATGCGCGATCA / 5 Phos / TCGTGCGCGATCATGCGCGATCA / 5 Phos / GTCATGATCGCGCATGATCGCGCA TGCGGAGTGATGCGGAGTGA / 5 Phos /TTCGTGCGGAGTGATGCGGAGTGA / 5 Phos / GTCATCACTCCGCATCACTCCGCA TGCGGATTCCTGCGGATTCC / 5 Phos / TCGTGCGGATTCCTGCGGATTCC / 5Phos / GTCAGGAATCCGCAGGAATCCGCA TGCGGCTACATGCGGCTACA / 5 Phos /TTCGTGCGGCTACATGCGGCTACA / 5 Phos / GTCATGTAGCCGCATGTAGCCGCA TGCGTAACAATGCGTAACAA / 5 Phos / TCGTGCGTAACAATGCGTAACAA / 5Phos/GTCATTGTTACGCATTGTTACGCA TGCGTCCTCATGCGTCCTCA / 5 Phos / TTCGTGCGTCCTCATGCGTCCTCA / 5 Phos /GTCATGAGGACGCATGAGGACGCA TGCGTTCAGCTGCGTTCAGC / 5 Phos / TCGTGCGTTCAGCTGCGTTCAGC / 5 Phos /GTCAGCTGAACGCAGCTGAACGCA TGCTTCAGCGTGCTTCAGCG / 5 Phos / TCGTGCTTCAGCGTGCTTCAGCG / 5 Phos /GTCACGCTGAAGCACGCTGAAGCA TGCTTGCCTCTGCTTGCCTC / 5 Phos /TTCGTGCTTGCCTCTGCTTGCCTC /5Phos /GTCAGAGGCAAGCAGAGGCAAGCA TGGAACTGGCTGGAACTGGC / 5 Phos / TCGTGGAACTGGCTGGAACTGGC / 5 Pho s / GTCAGCCAGTTCCAGCCAGTTCCA TGTATTGGAGTGTATTGGAG / 5 Phos / TCGTGTATTGGAGTGTATTGGAG / 5 Pho s / GTCACTCCAATACACTCCAATACA TGTGGAACGGTGTGGAACGG / 5 Phos / TTCGTGTGGAACGGTGTGGAACGG /5Phos /GTCACCGTTCCACACCGTTCCACA TGTGGAGTCGTGTGGAGTCG / 5Phos / TTCGTGTGGAGTCGTGTGGAGTCG /5Phos /GTCACGACTCCACACGACTCCACA TGTGTGGCCATGTGTGGCCA / 5Phos / TCGTGTGTGGCCATGTGTGGCCA / 5 Pho s / GTCATGGCCACACATGGCCACACA TGTTAAGAGGTGTTAAGAGG / 5 Pho s / TCGTGTTAAGAGGTGTTAAGAGG / 5 Phos / GTCACCTCTTAACACCTCTTAACA TGTTACTCACTGTTACTCAC / 5 Pho s / TTCGTGTTACTCACTGTTACTCAC / 5 Phos / GTCAGTGAGTAACAGTGAGTAACA TGTTCGCAGCTGTTCGCAGC / 5 Pho s / TCGTGTTCGCAGCTGTTCGCAGC / 5 Phos /GTCAGCTGCGAACAGCTGCGAACA TGTTGGCATATGTTGGCATA / 5 Phos / TCGTGTTGGCATATGTTGGCATA / 5 Phos /GTCATATGCCAACATATGCCAACA TTAAGTGCAGTTAAGTGCAG / 5 Phos / TCGTTAAGTGCAGTTAAGTGCAG / 5 Phos /GTCACTGCACTTAACTGCACTTAA TTAGAGATCCTTAGAGATCC / 5 Phos /TTCGTTAGAGATCCTTAGAGATCC / 5 Phos /GTCAGGATCTCTAAGGATCTCTAA TTAGGTCAGATTAGGTCAGA / 5 Phos / TTCGTTAGGTCAGATTAGGTCAGA / 5 Phos /GTCATCTGACCTAATCTGACCTAA TTATCTCCTGTTATCTCCTG / 5 Phos /TTCGTTATCTCCTGTTATCTCCTG /5Phos /GTCACAGGAGATAACAGGAGATAA TTCAGTGTCGTTCAGTGTCG / 5 Phos /TTCGTTCAGTGTCGTTCAGTGTCG / 5 PhoS / GTCACGACACTGAACGACACTGAA TTCCAAGAAGTTCCAAGAAG /5Phos /TTCGTTCCAAGAAGTTCCAAGAAG / 5 Phos / GTCACTTCTTGGAACTTCTTGGAA TTCCACCGCATTCCACCGCA / 5Phos / TTCGTTCCACCGCATTCCACCGCA / 5Phos / GTCATGCGGTGGAATGCGGTGGAA TTCCACTATCTTCCACTATC / 5 Phos / TCGTTCCACTATCTTCCACTATC / 5 Phos / GTCAGATAGTGGAAGATAGTGGAA TTCCACTTGATTCCACTTGA / 5 Pho s / TTCGTTCCACTTGATTCCACTTGA / 5 Phos /GTCATCAAGTGGAATCAAGTGGAA TTCCGAGAGCTTCCGAGAGC / 5 Pho s / TCGTTCCGAGAGCTTCCGAGAGC / 5 Phos /GTCAGCTCTCGGAAGCTCTCGGAA TTCCTGAGCCTTCCTGAGCC / 5 Phos /TTCGTTCCTGAGCCTTCCTGAGCC / 5 Phos / GTCAGGCTCAGGAAGGCTCAGGAA TTCGAGTCGCTTCGAGTCGC / 5 Phos / TCGTTCGAGTCGCTTCGAGTCGC / 5 Phos /GTCAGCGACTCGAAGCGACTCGAA TTCGTGCCAGTTCGTGCCAG / 5 Phos /TTCGTTCGTGCCAGTTCGTGCCAG /5Phos /GTCACTGGCACGAACTGGCACGAA TTCTACACCATTCTACACCA / 5 Phos / TCGTTCTACACCATTCTACACCA / 5 Pho s / GTCATGGTGTAGAATGGTGTAGAA TTCTCGCTCCTTCTCGCTCC / 5Phos / TCGTTCTCGCTCCTTCTCGCTCC / 5 Pho s / GTCAGGAGCGAGAAGGAGCGAGAA TTCTGGCCACTTCTGGCCAC / 5 Phos / TTCGTTCTGGCCACTTCTGGCCAC / 5Phos /GTCAGTGGCCAGAAGTGGCCAGAA TTGAATGGTGTTGAATGGTG / 5 Phos / TTCGTTGAATGGTGTTGAATGGTG / 5 Phos /GTCACACCATTCAACACCATTCAA TTGAGCCGACTTGAGCCGAC / 5 Phos / TCGTTGAGCCGACTTGAGCCGAC / 5 Phos /GTCAGTCGGCTCAAGTCGGCTCAA TTGCGCCAAGTTGCGCCAAG / 5 Phos /TTCGTTGCGCCAAGTTGCGCCAAG / 5Phos/GTCACTTGGCGCAACTTGGCGCAA TTGCTCTTAGTTGCTCTTAG / 5 Phos / TCGTTGCTCTTAGTTGCTCTTAG / 5 Phos / GTCACTAAGAGCAACTAAGAGCAA TTGGCAAGGCTTGGCAAGGC / 5 Phos /TTCGTTGGCAAGGCTTGGCAAGGC / 5 Phos /GTCAGCCTTGCCAAGCCTTGCCAA TTGGCTCACGTTGGCTCACG / 5 Phos /TTCGTTGGCTCACGTTGGCTCACG / 5 Phos /GTCACGTGAGCCAACGTGAGCCAA TTGGCTTCCATTGGCTTCCA / 5 Phos /TTCGTTGGCTTCCATTGGCTTCCA / 5 Phos /GTCATGGAAGCCAATGGAAGCCAA TTGTGGATACTTGTGGATAC / 5 Phos /TTCGTTGTGGATACTTGTGGATAC / 5 Phos /GTCAGTATCCACAAGTATCCACAA AACACGGATGAACACGGATG / 5 Phos /TTCGAACACGGATGAACACGGATG /5Phos /GTCACATCCGTGTTCATCCGTGTT AACAGACCGGAACAGACCGG / 5 Phos / TTCGAACAGACCGGAACAGACCGG / 5 Phos /GTCACCGGTCTGTTCCGGTCTGTT AACAGTGATCAACAGTGATC / 5 Phos / TCGAACAGTGATCAACAGTGATC / 5 Pho s / GTCAGATCACTGTTGATCACTGTT AACCATCTTGAACCATCTTG / 5 Phos / TCGAACCATCTTGAACCATCTTG / 5 Phos / GTCACAAGATGGTTCAAGATGGTT AACGGTGACGAACGGTGACG / 5Phos / TCGAACGGTGACGAACGGTGACG / 5 Pho s / GTCACGTCACCGTTCGTCACCGTT AACTACGCGGAACTACGCGG /5Phos /TTCGAACTACGCGGAACTACGCGG / 5 Pho s / GTCACCGCGTAGTTCCGCGTAGTT AACTAGGCTTAACTAGGCTT / 5Phos / TCGAACTAGGCTTAACTAGGCTT / 5 Phos /GTCAAAGCCTAGTTAAGCCTAGTT AACTGGCGTGAACTGGCGTG / 5 Phos / TTCGAACTGGCGTGAACTGGCGTG / 5Phos /GTCACACGCCAGTTCACGCCAGTT AAGACAGGATAAGACAGGAT / 5 Phos / TCGAAGACAGGATAAGACAGGAT / 5 Phos / GTCAATCCTGTCTTATCCTGTCTT AAGAGCCAGTAAGAGCCAGT / 5 Phos / TCGAAGAGCCAGTAAGAGCCAGT / 5 Phos / GTCAACTGGCTCTTACTGGCTCTT AAGAGTATCGAAGAGTATCG / 5 Phos / TCGAAGAGTATCGAAGAGTATCG / 5 Phos / GTCACGATACTCTTCGATACTCTT AAGGAACACTAAGGAACACT / 5 Phos / TCGAAGGAACACTAAGGAACACT / 5 Phos /GTCAAGTGTTCCTTAGTGTTCCTT AAGTCGCAGGAAGTCGCAGG / 5 Phos / TCGAAGTCGCAGGAAGTCGCAGG /5Phos /GTCACCTGCGACTTCCTGCGACTT AATCACAGTCAATCACAGTC /5Phos/ TCGAATCACAGTCAATCACAGTC /5Phos/GTCAGACTGTGATTGACTGTGATT AATCGCCATTAATCGCCATT /5Phos/ TCGAATCGCCATTAATCGCCATT /5Phos/GTCAAATGGCGATTAATGGCGATT AATTGCGGCCAATTGCGGCC /5Phos/ TCGAATTGCGGCCAATTGCGGCC /5Phos/GTCAGGCCGCAATTGGCCGCAATT ACAACTCGCGACAACTCGCG /5Phos/TTCGACAACTCGCGACAACTCGCG /5Phos/GTCACGCGAGTTGTCGCGAGTTGT ACAAGCTGCGACAAGCTGCG /5Phos/TTCGACAAGCTGCGACAAGCTGCG /5Phos/GTCACGCAGCTTGTCGCAGCTTGT ACACCAATTCACACCAATTC /5Phos/TTCGACACCAATTCACACCAATTC /5Phos/GTCAGAATTGGTGTGAATTGGTGT ACACGAAGGTACACGAAGGT /5Phos/TTCGACACGAAGGTACACGAAGGT /5Phos/GTCAACCTTCGTGTACCTTCGTGT ACAGAATGTGACAGAATGTG /5Phos/TTCGACAGAATGTGACAGAATGTG /5Phos/GTCACACATTCTGTCACATTCTGT ACATAGTGGTACATAGTGGT /5Phos/ TCGACATAGTGGTACATAGTGGT /5Phos/GTCAACCACTATGTACCACTATGT ACCACCACGTACCACCACGT /5Phos/TTCGACCACCACGTACCACCACGT /5Phos/GTCAACGTGGTGGTACGTGGTGGT ACCGACAGCTACCGACAGCT /5Phos/ TCGACCGACAGCTACCGACAGCT /5Phos/GTCAAGCTGTCGGTAGCTGTCGGT ACCGGCTAGTACCGGCTAGT /5Phos/ TCGACCGGCTAGTACCGGCTAGT /5Phos/GTCAACTAGCCGGTACTAGCCGGT ACCGTAGATTACCGTAGATT /5Phos/ TCGACCGTAGATTACCGTAGATT /5Phos/GTCAAATCTACGGTAATCTACGGT ACCGTGACTTACCGTGACTT /5Phos/TTCGACCGTGACTTACCGTGACTT /5Phos/GTCAAAGTCACGGTAAGTCACGGT ACCTCACAACACCTCACAAC /5Phos/TTCGACCTCACAACACCTCACAAC /5Phos/GTCAGTTGTGAGGTGTTGTGAGGT ACCTTGTCCTACCTTGTCCT /5Phos/ TCGACCTTGTCCTACCTTGTCCT /5Phos/GTCAAGGACAAGGTAGGACAAGGT ACGAAGCAGGACGAAGCAGG /5Phos/ TCGACGAAGCAGGACGAAGCAGG /5Phos/GTCACCTGCTTCGTCCTGCTTCGT ACGACTGGACACGACTGGAC /5Phos/ TCGACGACTGGACACGACTGGAC /5Phos/GTCAGTCCAGTCGTGTCCAGTCGT ACGAGAGCAGACGAGAGCAG /5Phos/ TCGACGAGAGCAGACGAGAGCAG /5Phos/GTCACTGCTCTCGTCTGCTCTCGT ACGCCTGTTGACGCCTGTTG /5Phos/ TCGACGCCTGTTGACGCCTGTTG /5Phos/GTCACAACAGGCGTCAACAGGCGT ACGCGACTTCACGCGACTTC /5Phos/TTCGACGCGACTTCACGCGACTTC /5Phos/GTCAGAAGTCGCGTGAAGTCGCGT ACGGATTGATACGGATTGAT /5Phos/ TCGACGGATTGATACGGATTGAT /5Phos/GTCAATCAATCCGTATCAATCCGT ACGGCTCATGACGGCTCATG /5Phos/TTCGACGGCTCATGACGGCTCATG /5Phos/GTCACATGAGCCGTCATGAGCCGT ACGGTAAGATACGGTAAGAT /5Phos/TTCGACGGTAAGATACGGTAAGAT /5Phos/GTCAATCTTACCGTATCTTACCGT ACGGTAGCACACGGTAGCAC /5Phos/ TCGACGGTAGCACACGGTAGCAC /5Phos/GTCAGTGCTACCGTGTGCTACCGT ACGGTGTTCGACGGTGTTCG /5Phos/TTCGACGGTGTTCGACGGTGTTCG /5Phos/GTCACGAACACCGTCGAACACCGT ACGTCGATGGACGTCGATGG /5Phos/TTCGACGTCGATGGACGTCGATGG /5Phos/GTCACCATCGACGTCCATCGACGT ACGTGCAGACACGTGCAGAC /5Phos/TTCGACGTGCAGACACGTGCAGAC /5PhoS/GTCAGTCTGCACGTGTCTGCACGT ACGTTCCAGCACGTTCCAGC /5Phos/TTCGACGTTCCAGCACGTTCCAGC /5Phos/GTCAGCTGGAACGTGCTGGAACGT ACTAGCTTGTACTAGCTTGT /5Phos/TTCGACTAGCTTGTACTAGCTTGT /5Phos/GTCAACAAGCTAGTACAAGCTAGT ACTAGGACGCACTAGGACGC /5Phos/TTCGACTAGGACGCACTAGGACGC /5Phos/GTCAGCGTCCTAGTGCGTCCTAGT ACTCACCTGGACTCACCTGG /5Phos/TTCGACTCACCTGGACTCACCTGG /5Phos/GTCACCAGGTGAGTCCAGGTGAGT ACTCATATCGACTCATATCG /5Phos/TTCGACTCATATCGACTCATATCG /5Phos/GTCACGATATGAGTCGATATGAGT ACTGACCGTGACTGACCGTG /5Phos/TTCGACTGACCGTGACTGACCGTG /5Phos/GTCACACGGTCAGTCACGGTCAGT ACTTCTAACCACTTCTAACC /5Phos/TTCGACTTCTAACCACTTCTAACC /SPhos/GTCAGGTTAGAAGTGGTTAGAAGT
ACTTGGCGCTACTTGGCGCT /5Phos/TGACACTTGGCGCTACTTGGCGCT /5Phos/GGGAAGCGCCAAGTAGCGCCAAGT AGAACGCTCCAGAACGCTCC /5Phos/TGACAGAACGCTCCAGAACGCTCC /5Phos/GGGAGGAGCGTTCTGGAGCGTTCT AGAACTTAGGAGAACTTAGG /5Phos/ GACAGAACTTAGGAGAACTTAGG /5Phos/GGGACCTAAGTTCTCCTAAGTTCT AGAAGCGCATAGAAGCGCAT /5Phos/ GACAGAAGCGCATAGAAGCGCAT /5Phos/GGGAATGCGCTTCTATGCGCTTCT AGACAATAGCAGACAATAGC /5PhoS/ GACAGACAATAGCAGACAATAGC /5Phos/GGGAGCTATTGTCTGCTATTGTCT AGACCGAGACAGACCGAGAC /5PhoS/TGACAGACCGAGACAGACCGAGAC /5Phos/GGGAGTCTCGGTCTGTCTCGGTCT AGACGCTGTCAGACGCTGTC /5Phos/TGACAGACGCTGTCAGACGCTGTC /5Phos/GGGAGACAGCGTCTGACAGCGTCT AGACGTAGCGAGACGTAGCG /5Phos/TGACAGACGTAGCGAGACGTAGCG /5Phos/GGGACGCTACGTCTCGCTACGTCT AGAGAGCTCTAGAGAGCTCT /5Phos/TGACAGAGAGCTCTAGAGAGCTCT /5Phos/GGGAAGAGCTCTCTAGAGCTCTCT AGAGGCCACTAGAGGCCACT /5Phos/TGACAGAGGCCACTAGAGGCCACT /5Phos/GGGAAGTGGCCTCTAGTGGCCTCT AGATTGCCGCAGATTGCCGC /5Phos/TGACAGATTGCCGCAGATTGCCGC /5Phos/GGGAGCGGCAATCTGCGGCAATCT AGCACGATGCAGCACGATGC /5Phos/TGACAGCACGATGCAGCACGATGC /5Phos/GGGAGCATCGTGCTGCATCGTGCT AGCAGAGAATAGCAGAGAAT /5Phos/ GACAGCAGAGAATAGCAGAGAAT /5Phos/GGGAATTCTCTGCTATTCTCTGCT AGCCACAAGGAGCCACAAGG /5Phos/ GACAGCCACAAGGAGCCACAAGG /5Phos/GGGACCTTGTGGCTCCTTGTGGCT AGCCAGGAAGAGCCAGGAAG /5Phos/TGACAGCCAGGAAGAGCCAGGAAG /5Phos/GGGACTTCCTGGCTCTTCCTGGCT AGCTCTGGAGAGCTCTGGAG /5Phos/TGACAGCTCTGGAGAGCTCTGGAG /5Phos/GGGACTCCAGAGCTCTCCAGAGCT AGCTTGGCAGAGCTTGGCAG /5Phos/TGACAGCTTGGCAGAGCTTGGCAG /5Phos/GGGACTGCCAAGCTCTGCCAAGCT AGGAATAACGAGGAATAACG /5Phos/TGACAGGAATAACGAGGAATAACG /5Phos/GGGACGTTATTCCTCGTTATTCCT AGGAATTGACAGGAATTGAC /5Phos/TGACAGGAATTGACAGGAATTGAC /5Phos/GGGAGTCAATTCCTGTCAATTCCT AGGAGGAATTAGGAGGAATT /5Phos/TGACAGGAGGAATTAGGAGGAATT /5Phos/GGGAAATTCCTCCTAATTCCTCCT AGGATAGGCCAGGATAGGCC /5Phos/TGACAGGATAGGCCAGGATAGGCC /5Phos/GGGAGGCCTATCCTGGCCTATCCT AGGTGGCCTTAGGTGGCCTT /5Phos/ GACAGGTGGCCTTAGGTGGCCTT /5Phos/GGGAAAGGCCACCTAAGGCCACCT AGGTGTTGCGAGGTGTTGCG /5Phos/ GACAGGTGTTGCGAGGTGTTGCG /5Phos/GGGACGCAACACCTCGCAACACCT AGGTTAGGTGAGGTTAGGTG /5Phos/TGACAGGTTAGGTGAGGTTAGGTG /5Phos/GGGACACCTAACCTCACCTAACCT AGTCCGTCCTAGTCCGTCCT /5Phos/TGACAGTCCGTCCTAGTCCGTCCT /5Phos/GGGAAGGACGGACTAGGACGGACT AGTCGATCCGAGTCGATCCG /5Phos/TGACAGTCGATCCGAGTCGATCCG /5Phos/GGGACGGATCGACTCGGATCGACT AGTCGC TGCTAGTCGC TGC T / 5Phos/TGACAGTCGCTGCTAGTCGCTGCT / 5 Pho s / GGGAAGCAGCGACTAGCAGCGACT AGTCGTCCTCAGTCGTCCTC / 5Phos /TGACAGTCGTCCTCAGTCGTCCTC / 5 Phos /GGGAGAGGACGACTGAGGACGACT AGTGTTCCGTAGTGTTCCGT / 5 Phos / GACAGTGTTCCGTAGTGTTCCGT / 5 Phos /GGGAACGGAACACTACGGAACACT AGTTGCTCATAGTTGCTCAT / 5Phos/TGACAGTTGCTCATAGTTGCTCAT / 5 Phos /GGGAATGAGCAACTATGAGCAACT ATAACGTGAGATAACGTGAG /5Phos / GACATAACGTGAGATAACGTGAG / 5 Pho s /GGGACTCACGTTATCTCACGTTAT ATACGCAGGCATACGCAGGC / 5 PhoS / GACATACGCAGGCATACGCAGGC / 5 Pho s / GGGAGCCTGCGTATGCCTGCGTAT ATACTGATGCATACTGATGC / 5 PhoS / TGAC ATAC TGATGC ATACTGATGC / 5 Phos / GGGAGCATCAGTATGCATCAGTAT ATAGTTCGTCATAGTTCGTC / 5 Phos / GACATAGTTCGTCATAGTTCGTC / 5 Phos /GGGAGACGAACTATGACGAACTAT ATATCTTCGCATATCTTCGC / 5 Phos / GACATATCTTCGCATATCTTCGC / 5 Phos /GGGAGCGAAGATATGCGAAGATAT ATATGCCTTCATATGCCTTC / 5 Phos /TGACATATGCCTTCATATGCCTTC / 5 Pho s / GGGAGAAGGC ATATGAAGGCATAT ATCAGATCACATCAGATCAC / 5 Phos / GACATCAGATCACATCAGATCAC / 5 Phos /GGGAGTGATCTGATGTGATCTGAT ATCCAATCTGATCCAATCTG / 5 Phos / TGACATCCAATCTGATCCAATCTG / 5Phos / GGGAC AGATTGGATC AGATTGGAT ATCCACAGCGATCCACAGCG /5Phos /TGACATCCACAGCGATCCACAGCG / 5Phos /GGGACGCTGTGGATCGCTGTGGAT ATCCGGAACGATCCGGAACG / 5 Phos /TGACATCCGGAACGATCCGGAACG / 5 PhoS / GGGACGTTCCGGATCGTTCCGGAT ATCGGCTTCCATCGGCTTCC / 5 Phos /TGACATCGGCTTCCATCGGCTTCC / 5 Phos /GGGAGGAAGCCGATGGAAGCCGAT ATCGTCGGAGATCGTCGGAG / 5 Phos / TGACATCGTCGGAGATCGTCGGAG / 5 Phos /GGGACTCCGACGATCTCCGACGAT ATCTCTCACGATCTCTCACG / 5 Pho s / GACATC CTCACGATCTCTCACG / 5Phos/GGGACGTGAGAGATCGTGAGAGAT ATGCACCTGCATGCACCTGC / 5Phos /TGACATGCACCTGCATGCACCTGC / 5 Pho s / GGGAGCAGGTGCATGCAGGTGCAT ATGCAGTCGCATGCAGTCGC / 5 Phos / GACATGCAGTCGCATGCAGTCGC / 5 Phos /GGGAGCGACTGCATGCGACTGCAT ATGCCGTAGGATGCCGTAGG / 5Phos/TGACATGCCGTAGGATGCCGTAGG / 5 Phos /GGGACCTACGGCATCCTACGGCAT ATGCGCGATCATGCGCGATC /5Phos /TGACATGCGCGATCATGCqCGATC / 5 Phos /GGGAGATCGCGCATGATCGCGCAT ATGTGGTGATATGTGGTGAT / 5 Pho s / TGAC ATGTGGTGATATGTGGTGAT / 5Phos / GGGAATCACCACATATCACCACAT ATTACGAGCCATTACGAGCC / 5Phos/TGACATTACGAGCCATTACGAGCC /5Phos /GGGAGGCTCGTAATGGCTCGTAAT ATTCCACGGCATTCCACGGC / 5 Phos / TGACATTCCACGGCATTCCACGGC / 5 Phos / GGGAGCCGTGGAATGCCGTGGAAT ATTCGGCGTCATTCGGCGTC / 5 Phos / TGACATTCGGCGTCATTCGGCGTC / 5 Pho s / GGGAGACGCCGAATGACGCCGAAT ATTGGAAGCCATTGGAAGCC /5Phos /TGACATTGGAAGCCATTGGAAGCC / 5 Phos /GGGAGGCTTCCAATGGCTTCCAAT ATTGTCGGCCATTGTCGGCC / 5Phos/TGACATTGTCGGCCATTGTCGGCC / 5 Phos / GGGAGGCCGACAATGGCCGACAAT CAACCGCTTGCAACCGCTTG / 5 Phos / GACCAACCGCTTGCAACCGCTTG / 5 Phos / GGGACAAGCGGTTGCAAGCGGTTG CAACTGGTGGCAACTGGTGG / 5 PhoS / TGACCAACTGGTGGCAACTGGTGG / 5Phos / GGGACCACCAGTTGCCACCAGTTG CAAGATGGTGCAAGATGGTG / 5Phos / TGACCAAGATGGTGCAAGATGGTG / 5 Phos / GGGACACCATCTTGCACCATCTTG CAAGATTCGACAAGATTCGA / 5 Phos /TGACCAAGATTCGACAAGATTCGA / 5 Phos /GGGATCGAATCTTGTCGAATCTTG CAAGCACGAGCAAGCACGAG / 5 Phos / GACCAAGCACGAGCAAGCACGAG / 5Phos /GGGACTCGTGCTTGCTCGTGCTTG CAAGCCTGTGCAAGCCTGTG / 5 Phos / TGACCAAGCCTGTGCAAGCCTGTG / 5 Pho s / GGGACACAGGCTTGCACAGGCTTG CAAGCTCACGCAAGCTCACG / 5 Phos /TGACCAAGCTCACGCAAGCTCACG / 5 Phos / GGGACGTGAGCTTGCGTGAGCTTG CAAGGTTGCGCAAGGTTGCG / 5 Phos / TGACCAAGGTTGCGCAAGGTTGCG / 5 Phos /GGGACGCAACCTTGCGCAACCTTG CAAGTCGACGCAAGTCGACG / 5 Phos / GACCAAGTCGACGCAAGTCGACG / 5Phos /GGGACGTCGACTTGCGTCGACTTG CACCACGAAGCACCACGAAG / 5 Pho s / GACCACCACGAAGCACCACGAAG / 5 Phos / GGGACTTCGTGGTGCTTCGTGGTG CACCGATATTCACCGATATT / 5Phos / TGACCACCGATATTCACCGATATT / 5 Phos / GGGAAATATCGGTGAATATCGGTG CACCGTCGAACACCGTCGAA / 5 Phos / TGACCACCGTCGAACACCGTCGAA / 5 Phos / GGGATTCGACGGTGTTCGACGGTG CACCGTGACACACCGTGACA / 5 Phos / TGACCACCGTGACACACCGTGACA / 5 Phos /GGGATGTCACGGTGTGTCACGGTG CACCTGCTGACACCTGCTGA / 5 Pho s / TGACCACCTGCTGACACCTGCTGA / 5 Pho s / GGGATCAGC AGGTGTC AGC AGGTG CACGCACATACACGCACATA / 5Phos / TGACCACGCACATACACGCACATA / 5 Pho s / GGGATATGTGCGTGTATGTGCGTG CACGCTAAGGCACGCTAAGG / 5 Phos / TGACCACGCTAAGGCACGCTAAGG /5Phos /GGGACCTTAGCGTGCCTTAGCGTG CACGTAATCTCACGTAATCT / 5 Phos /TGACCACGTAATCTCACGTAATCT / 5 Phos /GGGAAGATTACGTGAGATTACGTG CACGTGGAGTCACGTGGAGT / 5 Phos / TGACCACGTGGAGTCACGTGGAGT / 5 Phos / GGGAACTCCACGTGACTCCACGTG CACTCGAGAGCACTCGAGAG / 5 PhoS / GACCACTCGAGAGCACTCGAGAG / 5Phos / GGGACTCTCGAGTGCTCTCGAGTG CACTCTCTGACACTCTCTGA / 5Phos / TGACCACTCTCTGACACTCTCTGA / 5 Pho s / GGGATCAGAGAGTGTCAGAGAGTG CACTCTGGCTCACTCTGGCT / 5 Phos / TGACCACTCTGGCTCACTCTGGCT / 5 Phos /GGGAAGCCAGAGTGAGCCAGAGTG CACTGCCATGCACTGCCATG / 5 Phos /TGACCACTGCCATGCACTGCCATG / 5 Phos / GGGACATGGCAGTGCATGGCAGTG CACTTGAACTCACTTGAACT / 5 PhoS / GACCACTTGAACTCACTTGAACT / 5 Phos / GGGAAGTTCAAGTGAGTTCAAGTG CAGACCTGAGCAGACCTGAG / 5Phos / TGACCAGACCTGAGCAGACCTGAG / 5 Pho s / GGGACTCAGGTCTGCTCAGGTCTG CAGCGAGCATCAGCGAGCAT / 5 Phos / TGACCAGCGAGCATCAGCGAGCAT / 5 Phos /GGGAATGCTCGC GATGC CGCTG CAGGAAGAGGCAGGAAGAGG / 5 Phos / GACCAGGAAGAGGCAGGAAGAGG / 5 Phos /GGGACCTCTTCCTGCCTCTTCCTG CAGTCTCATACAGTCTCATA / 5 Pho s / TGACCAGTCTCATACAGTCTCATA / 5 Phos / GGGATATGAGACTGTATGAGACTG CAGTTATCGACAGTTATCGA / 5 Pho s / TGACCAGTTATCGACAGTTATCGA / 5 Phos /GGGATCGATAACTGTCGATAACTG CATACACGCGCATACACGCG / 5 Phos / GACCATACACGCGCATACACGCG / 5Phos/GGGACGCGTGTATGCGCGTGTATG CATACCGACGCATACCGACG / 5 Phos / GACCATACCGACGCATACCGACG / 5 Phos / GGGACGTCGGTATGCGTCGGTATG CATCAATGGTCATCAATGGT /5Phos /TGACCATCAATGGTCATCAATGGT /5Phos /GGGAACCATTGATGACCATTGATG CATGACACCGCATGACACCG / 5Phos / TGACCATGACACCGCATGACACCG / 5 Phos /GGGACGGTGTCATGCGGTGTCATG CATGGTTCGGCATGGTTCGG / 5Phos/TGACCATGGTTCGGCATGGTTCGG / 5 Phos /GGGACCGAACCATGCCGAACCATG CATTGGAGCGCATTGGAGCG / 5 Phos / TGACCATTGGAGCGCATTGGAGCG /5Phos /GGGACGCTCCAATGCGCTCCAATG CCAACGAGAGCCAACGAGAG / 5 Phos / GACCCAACGAGAGCCAACGAGAG / 5 Phos /GGGACTCTCGTTGGCTCTCGTTGG CCAAGACCAGCCAAGACCAG /5Phos/ GACCCAAGACCAGCCAAGACCAG /5Phos/GGGACTGGTCTTGGCTGGTCTTGG CCAATCACGGCCAATCACGG /5PhoS/TGACCCAATCACGGCCAATCACGG /5Phos/GGGACCGTGATTGGCCGTGATTGG CCACCGTTGTCCACCGTTGT /5Phos/TGACCCACCGTTGTCCACCGTTGT /5Phos/GGGAACAACGGTGGACAACGGTGG CCAGATCGGACCAGATCGGA /5Phos/TGACCCAGATCGGACCAGATCGGA /5Phos/GGGATCCGATCTGGTCCGATCTGG CCGAAGTCAGCCGAAGTCAG /5Phos/TGACCCGAAGTCAGCCGAAGTCAG /5Phos/GGGACTGACTTCGGCTGACTTCGG CCGCTGAAGTCCGCTGAAGT /5Phos/ GACCCGCTGAAGTCCGCTGAAGT /5Phos/GGGAACTTCAGCGGACTTCAGCGG CCGGACCATACCGGACCATA /5Phos/TGACCCGGACCATACCGGACCATA /5Phos/GGGATATGGTCCGGTATGGTCCGG CCGTTCTAGGCCGTTCTAGG /5Phos/TGACCCGTTCTAGGCCGTTCTAGG /5PhoS/GGGACCTAGAACGGCCTAGAACGG CCTAATGCGGCCTAATGCGG /5Phos/TGACCCTAATGCGGCCTAATGCGG /5Phos/GGGACCGCATTAGGCCGCATTAGG CCTATGACGACCTATGACGA /5Phos/ GACCCTATGACGACCTATGACGA /5Phos/GGGATCGTCATAGGTCGTCATAGG CCTCACCAGTCCTCACCAGT /5Phos/TGACCCTCACCAGTCCTCACCAGT /5Phos/GGGAACTGGTGAGGAC GG GAGG CCTGAAGACGCCTGAAGACG /5Phos/TGACCCTGAAGACGCCTGAAGACG /5Phos/GGGACGTCTTCAGGCGTCTTCAGG CCTGACTCCTCCTGACTCCT /5Phos/TGACCCTGACTCCTCCTGACTCCT /5Phos/GGGAAGGAGTCAGGAGGAGTCAGG CGAAGAGTGGCGAAGAGTGG /5Phos/TGACCGAAGAGTGGCGAAGAGTGG /5Phos/GGGACCACTCTTCGCCACTCTTCG CGAAGGTGGTCGAAGGTGGT /5Phos/ GACCGAAGGTGGTCGAAGGTGGT /5Phos/GGGAACCACCTTCGACCACCTTCG CGACTAGCAGCGACTAGCAG /5Phos/ GACCGACTAGCAGCGACTAGCAG /5Phos/GGGACTGCTAGTCGCTGCTAGTCG CGACTCGAGACGACTCGAGA /5Phos/TGACCGACTCGAGACGACTCGAGA /5Phos/GGGATCTCGAGTCGTCTCGAGTCG CGACTTACAACGACTTACAA /5Phos/TGACCGACTTACAACGACTTACAA /5Phos/GGGATTGTAAGTCGTTGTAAGTCG CGAGGATTAACGAGGATTAA /5Phos/TGACCGAGGATTAACGAGGATTAA /5Phos/GGGATTAATCCTCGTTAATCCTCG CGAGGCATGTCGAGGCATGT /5Phos/TGACCGAGGCATGTCGAGGCATGT /5Phos/GGGAACATGCCTCGACATGCCTCG CGAGTCTGCTCGAGTCTGCT /5Phos/ GACCGAGTCTGCTCGAGTCTGCT /5Phos/GGGAAGCAGACTCGAGCAGACTCG CGAGTGAGCACGAGTGAGCA /5Phos/TGACCGAGTGAGCACGAGTGAGCA /5Phos/GGGATGCTCACTCGTGCTCACTCG CGATCGGAAGCGATCGGAAG /5Phos/TGACCGATCGGAAGCGATCGGAAG /5Phos/GGGACTTCCGATCGCTTCCGATCG CGATCTACCGCGATCTACCG /5Phos/TGACCGATCTACCGCGATCTACCG /5Phos/GGGACGGTAGATCGCGGTAGATCG CGCCAAGCTTCGCCAAGCTT /5Phos/TGACCGCCAAGCTTCGCCAAGCTT /5Phos/GGGAAAGCTTGGCGAAGCTTGGCG CGCCAGAATTCGCCAGAATT /5Phos/TGACCGCCAGAATTCGCCAGAATT /5Phos/GGGAAATTCTGGCGAATTCTGGCG CGCGAATGGACGCGAATGGA /5Phos/TGACCGCGAATGGACGCGAATGGA /5Phos/GGGATCCATTCGCGTCCATTCGCG CGCTCATCCTCGCTCATCCT /5Phos/ GACCGCTCATCCTCGCTCATCCT /5Phos/GGGAAGGATGAGCGAGGATGAGCG CGCTCGAATGCGCTCGAATG /5Phos/ GACCGCTCGAATGCGCTCGAATG /5Phos/GGGACATTCGAGCGCATTCGAGCG CGCTTACTATCGCTTACTAT /5Phos/TGACCGCTTACTATCGCTTACTAT /5Phos/GGGAATAGTAAGCGATAGTAAGCG CGCTTAGCGTCGCTTAGCGT /5Phos/TGACCGCTTAGCGTCGCTTAGCGT /5Phos/GGGAACGCTAAGCGACGCTAAGCG CGGACTTAAGCGGACTTAAG /5Phos/ GACCGGACTTAAGCGGACTTAAG /5Phos/GGGACTTAAGTCCGCTTAAGTCCG CGGCGAACAACGGCGAACAA /5Phos/TGACCGGCGAACAACGGCGAACAA /5Phos/GGGATTGTTCGCCGTTGTTCGCCG CGGCTTGGAACGGCTTGGAA /5Phos/ GACCGGCTTGGAACGGCTTGGAA /5Phos/GGGATTCCAAGCCGTTCCAAGCCG CGGTAGTGCTCGGTAGTGCT /5Phos/ GACCGGTAGTGCTCGGTAGTGCT /5Phos/GGGAAGCACTACCGAGCACTACCG CGGTTACACACGGTTACACA /5Phos/TGACCGGTTACACACGGTTACACA /5Phos/GGGATGTGTAACCGTGTGTAACCG CGTACCGTGTCGTACCGTGT /5Phos/TGACCGTACCGTGTCGTACCGTGT /5Phos/GGGAACACGGTACGACACGGTACG CGTAGAGCCACGTAGAGCCA /5Phos/TGACCGTAGAGCCACGTAGAGCCA /5Phos/GGGATGGCTCTACGTGGCTCTACG CGTAGGACTGCGTAGGACTG /5Phos/TGACCGTAGGACTGCGTAGGACTG /5Phos/GGGACAGTCCTACGCAGTCCTACG CGTATCACAACGTATCACAA /5Phos/ GACCGTATCACAACGTATCACAA /5Phos/GGGATTGTGATACGTTGTGATACG CGTGTTGCGACGTGTTGCGA /5Phos/ GACCGTGTTGCGACGTGTTGCGA /5Phos/GGGATCGCAACACGTCGCAACACG CGTTGGTCCACGTTGGTCCA /5Phos/ GACCGTTGGTCCACGTTGGTCCA /5Phos/GGGATGGACCAACGTGGACCAACG CTACAGCCGACTACAGCCGA /5Phos/ GACCTACAGCCGACTACAGCCGA /5Phos/GGGATCGGCTGTAGTCGGCTGTAG CTACGCAAGGCTACGCAAGG /5Phos/ GACCTACGCAAGGCTACGCAAGG /5Phos/GGGACCTTGCGTAGCCTTGCGTAG CTACGGTGTGCTACGGTGTG /5Phos/ GACCTACGGTGTGCTACGGTGTG /5Phos/GGGACACACCGTAGCACACCGTAG CTACGTTCCTCTACGTTCCT /5Phos/ GACCTACGTTCCTCTACGTTCCT /5Phos/GGGAAGGAACGTAGAGGAACGTAG CTAGAGGCAGCTAGAGGCAG /5Phos/TGACCTAGAGGCAGCTAGAGGCAG /5Phos/GGGACTGCCTCTAGCTGCCTCTAG CTAGGTCCAGCTAGGTCCAG /5Phos/TGACCTAGGTCCAGCTAGGTCCAG /5Phos/GGGACTGGACCTAGCTGGACCTAG CTAGGTCGCTCTAGGTCGCT /5Phos/ GACCTAGGTCGCTCTAGGTCGCT /5Phos/GGGAAGCGACCTAGAGCGACCTAG CTATCGCCGTCTATCGCCGT /5Phos/ GACCTATCGCCGTCTATCGCCGT /5Phos/GGGAACGGCGATAGACGGCGATAG CTATGGATCTCTATGGATCT /5Phos/TGACCTATGGATCTCTATGGATCT /5Phos/GGGAAGATCCATAGAGATCCATAG CTCGCGAGTTCTCGCGAGTT /5Phos/TGACCTCGCGAGTTCTCGCGAGTT /5Phos/GGGAAACTCGCGAGAACTCGCGAG CTCGTGGCAACTCGTGGCAA /5Phos/ GACCTCGTGGCAACTCGTGGCAA /5Phos/GGGATTGCCACGAGTTGCCACGAG CTCTACAACTCTCTACAACT /5Phos/ GACCTCTACAACTCTCTACAACT /5Phos/GGGAAGTTGTAGAGAGTTGTAGAG CTCTATATCGCTCTATATCG /5Phos/ GACCTCTATATCGCTCTATATCG /5Phos/GGGACGATATAGAGCGATATAGAG CTCTCCTTCACTCTCCTTCA /5Phos/TGACCTCTCCTTCACTCTCCTTCA /5PhoS/GGGATGAAGGAGAGTGAAGGAGAG CTCTCTTGCGCTCTCTTGCG /5Phos/TGACCTCTCTTGCGCTCTCTTGCG /5Phos/GGGACGCAAGAGAGCGCAAGAGAG CTCTGCGTTGCTCTGCGTTG /5Phos/TGACCTCTGCGTTGCTCTGCGTTG /5Phos/GGGACAACGCAGAGCAACGCAGAG CTGAATCCAGCTGAATCCAG /5Phos/TGACCTGAATCCAGCTGAATCCAG /5Phos/GGGAC GGATTCAGCTGGATTCAG CTGAGCTTGGCTGAGCTTGG /5Phos/TGACCTGAGCTTGGCTGAGCTTGG /5Phos/GGGACCAAGCTCAGCCAAGCTCAG CTGGATCCGACTGGATCCGA /5Phos/TGACCTGGATCCGACTGGATCCGA /5Phos/GGGATCGGATCCAGTCGGATCCAG CTGGTCTGATCTGGTCTGAT /5Phos/TGACCTGGTCTGATCTGGTCTGAT /5Phos/GGGAATCAGACCAGATCAGACCAG CTGTCCACAGCTGTCCACAG /5Phos/ GACCTGTCCACAGCTGTCCACAG /5PhoS/GGGACTGTGGACAGCTGTGGACAG CTGTCCTCCTCTGTCCTCCT /5Phos/TGACCTGTCCTCCTCTGTCCTCCT /5Phos/GGGAAGGAGGACAGAGGAGGACAG CTGTCGGATGCTGTCGGATG /5Phos/ GACCTGTCGGATGCTGTCGGATG /5Phos/GGGACATCCGACAGCATCCGACAG CTTCATCTGACTTCATCTGA /5Phos/ GACCTTCATCTGACTTCATCTGA /5PhoS/GGGATCAGATGAAGTCAGATGAAG CTTCCTGCGTCTTCCTGCGT /5Phos/ GACCTTCCTGCGTCTTCCTGCGT /5Phos/GGGAACGCAGGAAGACGCAGGAAG CTTCGGCTAGCTTCGGCTAG /5Phos/TGACCTTCGGCTAGCTTCGGCTAG /5Phos/GGGACTAGCCGAAGCTAGCCGAAG CTTCTTATGGCTTCTTATGG /5Phos/TGACCTTCTTATGGCTTCTTATGG /5PhoS/GGGACCATAAGAAGCCATAAGAAG CTTCTTGGATCTTCTTGGAT /5Phos/TGACCTTCTTGGATCTTCTTGGAT /5PhoS/GGGAATCCAAGAAGATCCAAGAAG CTTGCGATGGCTTGCGATGG /5Phos/ GACCTTGCGATGGCTTGCGATGG /5Phos/GGGACCATCGCAAGCCATCGCAAG GAACCTCAGCGAACCTCAGC /5Phos/TGACGAACCTCAGCGAACCTCAGC /5Phos/GGGAGCTGAGGTTCGCTGAGGTTC GAACGGATTAGAACGGATTA /5Phos/ GACGAACGGATTAGAACGGATTA /5Phos/GGGATAATCCGTTCTAATCCGTTC GAACGTCATTGAACGTCATT /5Phos/TGACGAACGTCATTGAACGTCATT /5Phos/GGGAAATGACGTTCAATGACGTTC GAACTGATCCGAACTGATCC /5Phos/ GACGAACTGATCCGAACTGATCC /5Phos/GGGAGGATCAGTTCGGATCAGTTC GACAGCAGTCGACAGCAGTC /5Phos/TGACGACAGCAGTCGACAGCAGTC /5Phos/GGGAGACTGCTGTCGACTGCTGTC GACCGAATGTGACCGAATGT /5Phos/ GACGACCGAATGTGACCGAATGT /5Phos/GGGAACATTCGGTCACATTCGGTC GACGCCATCAGACGCCATCA /5Phos/TGACGACGCCATCAGACGCCATCA /5Phos/GGGATGATGGCGTCTGATGGCGTC GACGCGATACGACGCGATAC /5Phos/ GACGACGCGATACGACGCGATAC /5Phos/GGGAGTATCGCGTCGTATCGCGTC GACGCTGTGAGACGCTGTGA /5Phos/TGACGACGCTGTGAGACGCTGTGA /5Phos/GGGATCACAGCGTCTCACAGCGTC GACGGACCTTGACGGACCTT /5Phos/ GACGACGGACCTTGACGGACCTT /5Phos/GGGAAAGGTCCGTCAAGGTCCGTC GACGGAGTCTGACGGAGTCT /5Phos/TGACGACGGAGTCTGACGGAGTCT /5Phos/GGGAAGACTCCGTCAGACTCCGTC GAGCACAACCGAGCACAACC /5Phos/TGACGAGCACAACCGAGCACAACC /5Phos/GGGAGGTTGTGCTCGGTTGTGCTC GAGGAAGACCGAGGAAGACC /5Phos/TGACGAGGAAGACCGAGGAAGACC /5Phos/GGGAGGTCTTCCTCGGTCTTCCTC GAGGCACGATGAGGCACGAT /5Phos/ GACGAGGCACGATGAGGCACGAT /5Phos/GGGAATCGTGCCTCATCGTGCCTC GAGGTGAAGCGAGGTGAAGC /5Phos/ GACGAGGTGAAGCGAGGTGAAGC /5Phos/GGGAGCTTCACCTCGCTTCACCTC GAGTCACACCGAGTCACACC /5Phos/ GACGAGTCACACCGAGTCACACC /5Phos/GGGAGGTGTGACTCGGTGTGACTC GAGTCCAGACGAGTCCAGAC /5Phos/TGACGAGTCCAGACGAGTCCAGAC /5Phos/GGGAGTCTGGACTCGTCTGGACTC GATAACCTGTGATAACCTGT /5Phos/TGACGATAACCTGTGATAACCTGT /5Phos/GGGAACAGGTTATCACAGGTTATC GATCCAAGGCGATCCAAGGC /5Phos/TGACGATCCAAGGCGATCCAAGGC /5Phos/GGGAGCCTTGGATCGCCTTGGATC GATCGAGCCAGATCGAGCCA /5Phos/TGACGATCGAGCCAGATCGAGCCA /5Phos/GGGATGGCTCGATCTGGCTCGATC GATGGCAATCGATGGCAATC /5Phos/TGACGATGGCAATCGATGGCAATC /5Phos/GGGAGATTGCCATCGATTGCCATC GATTACCAACGATTACCAAC /5Phos/TGACGATTACCAACGATTACCAAC /5Phos/GGGAGTTGGTAATCGTTGGTAATC GATTCGTCTTGATTCGTCTT /5Phos/TGACGATTCGTCTTGATTCGTCTT /5Phos/GGGAAAGACGAATCAAGACGAATC GATTGTTGGAGATTGTTGGA /5Phos/TGACGATTGTTGGAGATTGTTGGA /5Phos/GGGATCCAACAATCTCCAACAATC GCAACACGGAGCAACACGGA /5Phos/ GACGCAACACGGAGCAACACGGA /5Phos/GGGATCCGTGTTGCTCCGTGTTGC GCAATGGTACGCAATGGTAC /5Phos/TGACGCAATGGTACGCAATGGTAC /5Phos/GGGAGTACCATTGCGTACCATTGC GCACCACATTGCACCACATT /5Phos/ GACGCACCACATTGCACCACATT /5Phos/GGGAAATGTGGTGCAATGTGGTGC GCAGATTGGCGCAGATTGGC /5Phos/ GACGCAGATTGGCGCAGATTGGC /5Phos/GGGAGCCAATCTGCGCCAATCTGC GCAGGATAGCGCAGGATAGC /5Phos/TGACGCAGGATAGCGCAGGATAGC /5Phos/GGGAGCTATCCTGCGCTATCCTGC GCATTAATCCGCATTAATCC /5Phos/ GACGCATTAATCCGCATTAATCC /5PhoS/GGGAGGATTAATGCGGATTAATGC GCCACCATCTGCCACCATCT /5Phos/TGACGCCACCATCTGCCACCATCT /5Phos/GGGAAGATGGTGGCAGATGGTGGC GCCAGATACCGCCAGATACC /5Phos/TGACGCCAGATACCGCCAGATACC /5Phos/GGGAGGTATCTGGCGGTATCTGGC GCCATAGATAGCCATAGATA /5Phos/TGACGCCATAGATAGCCATAGATA /5Phos/GGGATATCTATGGCTATCTATGGC
GCCATTGGACGCCATTGGAC /5Phos/ CCCGCCATTGGACGCCATTGGAC /5Phos/GTTGGTCCAATGGCGTCCAATGGC GCCGAATCCTGCCGAATCCT /5Phos/TCCCGCCGAATCCTGCCGAATCCT /5Phos/GTTGAGGATTCGGCAGGATTCGGC GCCGTGTCTAGCCGTGTCTA /5Phos/ CCCGCCGTGTCTAGCCGTGTCTA /5Phos/GTTGTAGACACGGCTAGACACGGC GCCTTATCTCGCCTTATCTC /5Phos/TCCCGCCTTATCTCGCCTTATCTC /5Phos/GTTGGAGATAAGGCGAGATAAGGC GCCTTCGCTTGCCTTCGCTT /5Phos/TCCCGCCTTCGCTTGCCTTCGCTT /5Phos/GTTGAAGCGAAGGCAAGCGAAGGC GCGAAGTGGTGCGAAGTGGT /5Phos/ CCCGCGAAGTGGTGCGAAGTGGT /5Phos/GTTGACCACTTCGCACCACTTCGC GCGAATATCTGCGAATATCT /5Phos/TCCCGCGAATATCTGCGAATATCT /5Phos/GTTGAGATATTCGCAGATATTCGC GCGCTGATACGCGCTGATAC /5Phos/ CCCGCGCTGATACGCGCTGATAC /5Phos/GTTGGTATCAGCGCGTATCAGCGC GCGGACTTCTGCGGACTTCT /5Phos/TCCCGCGGACTTCTGCGGACTTCT /5Phos/GTTGAGAAGTCCGCAGAAGTCCGC GCGGATAAGTGCGGATAAGT /5Phos/ CCCGCGGATAAGTGCGGATAAGT /5Phos/GTTGACTTATCCGCACTTATCCGC GCGGTCCATTGCGGTCCATT /5Phos/ CCCGCGGTCCATTGCGGTCCATT /5Phos/GTTGAATGGACCGCAATGGACCGC GCTCAGTTAAGCTCAGTTAA /5Phos/TCCCGCTCAGTTAAGCTCAGTTAA /5Phos/GTTGTTAACTGAGCTTAACTGAGC GCTCATTCTAGCTCATTCTA /5Phos/TCCCGCTCATTCTAGCTCATTCTA /5Phos/GTTGTAGAATGAGCTAGAATGAGC GCTCCTAAGCGCTCCTAAGC /5Phos/ CCCGCTCCTAAGCGCTCCTAAGC /5Phos/GTTGGCTTAGGAGCGCTTAGGAGC GCTGGAAGGAGCTGGAAGGA /5Phos/TCCCGCTGGAAGGAGCTGGAAGGA /5Phos/GTTGTCCTTCCAGCTCCTTCCAGC GGAACAATGTGGAACAATGT /5Phos/ CCCGGAACAATGTGGAACAATGT /5Phos/GTTGACATTGTTCCACATTGTTCC GGAACATCTCGGAACATCTC /5Phos/ CCCGGAACATCTCGGAACATCTC /5Phos/GTTGGAGATGTTCCGAGATGTTCC GGAACCAGTAGGAACCAGTA /5Phos/TCCCGGAACCAGTAGGAACCAGTA /5Phos/GTTGTACTGGTTCCTACTGGTTCC GGAACGACTTGGAACGACTT /5Phos/TCCCGGAACGACTTGGAACGACTT /5Phos/GTTGAAGTCGTTCCAAGTCGTTCC GGAAGATGATGGAAGATGAT /5Phos/ CCCGGAAGATGATGGAAGATGAT /5Phos/GTTGATCATCTTCCATCATCTTCC GGAAGGAGACGGAAGGAGAC /5Phos/TCCCGGAAGGAGACGGAAGGAGAC /5Phos/GTTGGTCTCCTTCCGTCTCCTTCC GGAAGTGGTAGGAAGTGGTA /5Phos/ CCCGGAAGTGGTAGGAAGTGG A /5Phos/GTTGTACCACTTCCTACCACTTCC GGAATCAGGTGGAATCAGGT /5Phos/ CCCGGAATCAGGTGGAATCAGGT /5Phos/GTTGACCTGATTCCACCTGATTCC GGAATGTGTAGGAATGTGTA /5Phos/ CCCGGAATGTGTAGGAATGTGTA /5Phos/GTTGTACACATTCCTACACATTCC GGAGATAGGAGGAGATAGGA /5Phos/ CCCGGAGATAGGAGGAGATAGGA /5Phos/GT GTCCTATCTCCTCCTATCTCC GGAGCAATCCGGAGCAATCC /5Phos/TCCCGGAGCAATCCGGAGCAATCC /5Phos/GTTGGGATTGCTCCGGATTGC CC GGAGGACATCGGAGGACATC /5Phos/ CCCGGAGGACATCGGAGGACATC /5Phos/GTTGGATGTCCTCCGATGTCCTCC GGCAATAGCCGGCAATAGCC /5Phos/ CCCGGCAATAGCCGGCAATAGCC /5Phos/GTTGGGCTATTGCCGGCTATTGCC GGCAGAAGGAGGCAGAAGGA /5Phos/ CCCGGCAGAAGGAGGCAGAAGGA /5Phos/GTTGTCCTTCTGCCTCCTTCTGCC GGCAGGAATAGGCAGGAATA /5Phos/TCCCGGCAGGAATAGGCAGGAATA /5Phos/GTTGTATTCCTGCCTATTCCTGCC GGCATACACCGGCATACACC /5Phos/TCCCGGCATACACCGGCATACACC /5Phos/GTTGGGTGTATGCCGGTGTATGCC GGCCGTTGTAGGCCGTTGTA /5Phos/ CCCGGCCGTTGTAGGCCGTTGTA /5Phos/GTTGTACAACGGCCTACAACGGCC GGCCTGCTTAGGCCTGCTTA /5Phos/ CCCGGCCTGCTTAGGCCTGCTTA /5Phos/GTTGTAAGCAGGCCTAAGCAGGCC GGCGAGGTAAGGCGAGGTAA /5Phos/ CCCGGCGAGGTAAGGCGAGGTAA /5Phos/GTTGTTACCTCGCCTTACCTCGCC GGCGTGACATGGCGTGACAT /5Phos/TCCCGGCGTGACATGGCGTGACAT /5Phos/GTTGATGTCACGCCATGTCACGCC GGCTCCAAGAGGCTCCAAGA /5Phos/TCCCGGCTCCAAGAGGCTCCAAGA /5Phos/GTTGTCTTGGAGCCTCTTGGAGCC GGCTGCTCAAGGCTGCTCAA /5Phos/TCCCGGCTGCTCAAGGCTGCTCAA /5Phos/GTTGTTGAGCAGCCTTGAGCAGCC GGCTGTGCTTGGCTGTGCTT /5Phos/ CCCGGCTGTGCTTGGCTGTGCTT /5Phos/GTTGAAGCACAGCCAAGCACAGCC GGCTTCTGTCGGCTTCTGTC /5Phos/TCCCGGCTTCTGTCGGCTTCTGTC /5Phos/GTTGGACAGAAGCCGACAGAAGCC GGTATCGCTTGGTATCGCTT /5Phos/ CCCGGTATCGCTTGGTATCGCTT /5Phos/GTTGAAGCGATACCAAGCGATACC GGTCCTTCCAGGTCCTTCCA /5Phos/ CCCGGTCCTTCCAGGTCCTTCCA /5Phos/GTTGTGGAAGGACCTGGAAGGACC GGTCGGTTATGGTCGGTTAT /5Phos/ CCCGGTCGGTTATGGTCGGTTAT /5Phos/GTTGATAACCGACCATAACCGACC GGTCTGACGAGGTCTGACGA /5Phos/TCCCGGTCTGACGAGGTCTGACGA /5Phos/GTTGTCGTCAGACCTCGTCAGACC GGTGACCACTGGTGACCACT /5Phos/TCCCGGTGACCACTGGTGACCACT /5Phos/GTTGAGTGGTCACCAGTGGTCACC GGTGAGTCCAGGTGAGTCCA /5Phos/TCCCGGTGAGTCCAGGTGAGTCCA /5Phos/GTTGTGGACTCACCTGGACTCACC GGTGGCGAATGGTGGCGAAT /5Phos/TCCCGGTGGCGAATGGTGGCGAAT /5Phos/GTTGATTCGCCACCATTCGCCACC GGTGTATATCGGTGTATATC /5PhoS/ CCCGGTGTATATCGGTGTATATC /5Phos/GTTGGATATACACCGATATACACC GGTTCCTTAAGGTTCCTTAA /5Phos/TCCCGGTTCCTTAAGGTTCCTTAA /5Phos/GTTGTTAAGGAACCTTAAGGAACC GGTTCTACAAGGTTCTACAA /5Phos/TCCCGGTTCTACAAGGTTCTACAA /5Phos/GTTGTTGTAGAACCTTGTAGAACC GGTTGGCGTTGGTTGGCGTT /5Phos/ CCCGGTTGGCGTTGGTTGGCGTT /5Phos/GTTGAACGCCAACCAACGCCAACC GTAACACGCTGTAACACGCT /5Phos/TCCCGTAACACGCTGTAACACGCT /5Phos/GTTGAGCGTGTTACAGCGTGTTAC GTAGAGCGACGTAGAGCGAC /5Phos/TCCCGTAGAGCGACGTAGAGCGAC /5Phos/GTTGGTCGCTCTACGTCGCTCTAC GTAGCTGCTCGTAGCTGCTC /5Phos/ CCCGTAGCTGCTCGTAGCTGCTC /5Phos/GTTGGAGCAGCTACGAGCAGCTAC GTAGGTGGATGTAGGTGGAT /5Phos/ CCCGTAGGTGGATGTAGGTGGAT /5Phos/GTTGATCCACCTACATCCACCTAC GTAGTCAGCCGTAGTCAGCC /5Phos/TCCCGTAGTCAGCCGTAGTCAGCC /5Phos/GTTGGGCTGACTACGGCTGACTAC GTCCTTCCACGTCCTTCCAC /5Phos/TCCCGTCCTTCCACGTCCTTCCAC /5Phos/GTTGGTGGAAGGACGTGGAAGGAC GTCGAGCAGTGTCGAGCAGT /5Phos/TCCCGTCGAGCAGTGTCGAGCAGT /5Phos/GTTGACTGCTCGACACTGCTCGAC GTCGCACAGAGTCGCACAGA /5Phos/ CCCGTCGCACAGAGTCGCACAGA /5Phos/GTTGTCTGTGCGACTCTGTGCGAC GTCGCACTTCGTCGCACTTC /5Phos/TCCCGTCGCACTTCGTCGCACTTC /5Phos/GTTGGAAGTGCGACGAAGTGCGAC GTCTGGTGGTGTCTGGTGGT /5Phos/TCCCGTCTGGTGGTGTCTGG GGT /5Phos/GTTGACCACCAGACACCACCAGAC GTGAACTGTTGTGAACTGTT /5Phos/TCCCGTGAACTGTTGTGAACTGTT /5PhoS/GTTGAACAGTTCACAACAGTTCAC GTGCCATCCTGTGCCATCCT /5Phos/ CCCGTGCCATCCTGTGCCATCCT /5Phos/GTTGAGGATGGCACAGGATGGCAC GTGCGTGAACGTGCGTGAAC /5Phos/ CCCGTGCGTGAACGTGCGTGAAC /5Phos/GTTGGTTCACGCACGTTCACGCAC GTGGAGATGTGTGGAGATGT /5Phos/ CCCGTGGAGATGTGTGGAGATGT /5Phos/GTTGACATCTCCACACATCTCCAC GTGGTGCAACGTGGTGCAAC /5Phos/TCCCGTGGTGCAACGTGGTGCAAC /5Phos/GTTGGTTGCACCACGTTGCACCAC GTGTCGTTAAGTGTCGTTAA /5Phos/ CCCGTGTCGTTAAGTGTCGTTAA /5PhoS/GTTGTTAACGACACTTAACGACAC GTGTCTCAATGTGTCTCAAT /5Phos/TCCCGTGTCTCAATGTGTCTCAAT /5PhoS/GTTGATTGAGACACATTGAGACAC GTGTGATGACGTGTGATGAC /5Phos/TCCCGTGTGATGACGTGTGATGAC /5Phos/GTTGGTCATCACACGTCATCACAC GTTAGATCGCGTTAGATCGC /5Phos/ CCCGTTAGATCGCGTTAGATCGC /5Phos/GTTGGCGATCTAACGCGATCTAAC TAAGCCGATATAAGCCGATA /5Phos/ CCCTAAGCCGATATAAGCCGATA /5Phos/GTTGTATCGGCTTATATCGGCTTA TAAGGACACCTAAGGACACC /5Phos/ CCCTAAGGACACCTAAGGACACC /5Phos/GTTGGGTGTCCTTAGGTGTCCTTA TAAGGCCAAGTAAGGCCAAG /5Phos/ CCCTAAGGCCAAGTAAGGCCAAG /5Phos/GTTGCTTGGCCTTACTTGGCCTTA TAATAGCGAGTAATAGCGAG /5Phos/TCCCTAATAGCGAGTAATAGCGAG /5Phos/GTTGCTCGCTATTACTCGCTATTA TAATGGCCGGTAATGGCCGG /5Phos/ CCCTAATGGCCGGTAATGGCCGG /5Phos/GTTGCCGGCCATTACCGGCCATTA TACCATTGGATACCATTGGA /5Phos/ CCCTACCATTGGATACCATTGGA /5Phos/GTTGTCCAATGGTATCCAATGGTA TACGACCACCTACGACCACC /5Phos/ CCCTACGACCACCTACGACCACC /5Phos/GTTGGGTGGTCGTAGGTGGTCGTA TACGACCTTATACGACCTTA /5Phos/ CCCTACGACCTTATACGACCTTA /5Phos/GTTGTAAGGTCGTATAAGGTCGTA TACGTGATTCTACGTGATTC /5Phos/TCCCTACGTGATTCTACGTGATTC /5Phos/GTTGGAATCACGTAGAATCACGTA TACTAGTCAGTACTAGTCAG /5Phos/ CCCTACTAGTCAGTACTAGTCAG /5Phos/GTTGCTGACTAGTACTGACTAGTA TACTGACAAGTACTGACAAG /5Phos/ CCCTACTGACAAGTACTGACAAG /5Phos/GTTGCTTGTCAGTACTTGTCAGTA TACTGCTGGCTACTGCTGGC /5Phos/ CCCTACTGCTGGCTACTGCTGGC /5Phos/GTTGGCCAGCAGTAGCCAGCAGTA TAGACCGTAATAGACCGTAA /5Phos/ CCCTAGACCGTAATAGACCGTAA /5Phos/GTTGTTACGGTCTATTACGGTCTA TAGACGAAGATAGACGAAGA /5Phos/ CCCTAGACGAAGATAGACGAAGA /5Phos/GTTGTCTTCGTCTATCTTCGTCTA TAGCCTAGCCTAGCCTAGCC /5Phos/ CCCTAGCCTAGCCTAGCCTAGCC /5Phos/GTTGGGCTAGGCTAGGCTAGGCTA TAGCGAATTCTAGCGAATTC /5Phos/ CCCTAGCGAATTCTAGCGAATTC /5Phos/GTTGGAATTCGCTAGAATTCGCTA TAGCTGCCACTAGCTGCCAC /5Phos/ CCCTAGCTGCCACTAGCTGCCAC /5Phos/GTTGGTGGCAGCTAGTGGCAGCTA TAGGTAGGCATAGGTAGGCA /5Phos/TCCCTAGGTAGGCATAGGTAGGCA /5Phos/GTTGTGCCTACCTATGCCTACCTA TAGTCGTTACTAGTCGTTAC /5Phos/ CCCTAGTCGTTACTAGTCGTTAC /5Phos/GTTGGTAACGACTAGTAACGACTA TAGTGCGAAGTAGTGCGAAG /5Phos/ CCCTAGTGCGAAGTAGTGCGAAG /5Phos/GTTGCTTCGCACTACTTCGCACTA TAGTGGACGCTAGTGGACGC /5Phos/ CCCTAGTGGACGCTAGTGGACGC /5Phos/GTTGGCGTCCACTAGCGTCCACTA TATAACGGTGTATAACGGTG /5Phos/ CCCTATAACGGTGTATAACGGTG /5Phos/GTTGCACCGTTATACACCGTTATA TATAGAACCGTATAGAACCG /5Phos/TCCCTATAGAACCGTATAGAACCG /5Phos/GTTGCGGTTCTATACGGTTCTATA TATCCGAAGGTATCCGAAGG /5Phos/ CCCTATCCGAAGGTATCCGAAGG /5Phos/GTTGCCTTCGGATACCTTCGGATA TATCGGAGCCTATCGGAGCC /5Phos/TCCCTATCGGAGCCTATCGGAGCC /5Phos/GTTGGGCTCCGATAGGCTCCGATA TATCGGCCTGTATCGGCCTG /5Phos/TCCCTATCGGCCTGTATCGGCCTG /5Phos/GTTGCAGGCCGATACAGGCCGATA TATCGTCGGCTATCGTCGGC /5Phos/ CCCTATCGTCGGCTATCGTCGGC /5Phos/GTTGGCCGACGATAGCCGACGATA TATGCGCCACTATGCGCCAC /5Phos/ CCCTATGCGCCACTATGCGCCAC /5Phos/GTTGGTGGCGCATAGTGGCGCATA TATGGCCGTCTATGGCCGTC /5Phos/ CCCTATGGCCGTCTATGGCCGTC /5Phos/GTTGGACGGCCATAGACGGCCATA TATTCTTCCGTATTCTTCCG /5Phos/TCCCTATTCTTCCGTATTCTTCCG /5Phos/GTTGCGGAAGAATACGGAAGAATA TCAAGCAACGTCAAGCAACG /5Phos/ CCCTCAAGCAACGTCAAGCAACG /5Phos/GTTGCGTTGCTTGACGTTGCTTGA TCAAGCAGTCTCAAGCAGTC /5Phos/TCCCTCAAGCAGTCTCAAGCAGTC /5Phos/GTTGGACTGCTTGAGACTGCTTGA TCAAGTCCGATCAAGTCCGA /5Phos/TCCCTCAAGTCCGATCAAGTCCGA /5Phos/GTTGTCGGACTTGATCGGACTTGA TCAATCGAGATCAATCGAGA /5Phos/ CCCTCAATCGAGATCAATCGAGA /5Phos/GTTGTCTCGATTGATCTCGATTGA TCAATGTCGATCAATGTCGA /5Phos/ CCCTCAATGTCGATCAATGTCGA /5Phos/GTTGTCGACATTGATCGACATTGA TCACTGAGGCTCACTGAGGC /5Phos/TCCCTCACTGAGGCTCACTGAGGC /5Phos/GTTGGCCTCAGTGAGCCTCAGTGA TCAGCGAGACTCAGCGAGAC /5Phos/TCCCTCAGCGAGACTCAGCGAGAC /5Phos/GTTGGTCTCGCTGAGTCTCGCTGA TCAGGAGGAATCAGGAGGAA /5Phos/ CCCTCAGGAGGAATCAGGAGGAA /5Phos/GTTGTTCCTCCTGATTCCTCCTGA TCAGGCACAGTCAGGCACAG /5Phos/ CCCTCAGGCACAGTCAGGCACAG /5Phos/GTTGCTGTGCCTGACTGTGCCTGA TCAGGCTTCCTCAGGCTTCC /5Phos/TCCCTCAGGCTTCCTCAGGCTTCC /5Phos/GTTGGGAAGCCTGAGGAAGCCTGA TCCACACTCGTCCACACTCG /5Phos/TCCCTCCACACTCGTCCACACTCG /5Phos/GTTGCGAGTGTGGACGAGTGTGGA TCCACAGCGATCCACAGCGA /5Phos/ CCCTCCACAGCGATCCACAGCGA /5Phos/GTTGTCGCTGTGGATCGCTGTGGA TCCAGCTGCATCCAGCTGCA /5Phos/TCCCTCCAGCTGCATCCAGCTGCA /5Phos/GTTGTGCAGCTGGATGCAGCTGGA TCCATAATCCTCCATAATCC /5Phos/TCCCTCCATAATCCTCCATAATCC /5Phos/GTTGGGATTATGGAGGATTATGGA TCCGGACCAATCCGGACCAA /5Phos/ CCCTCCGGACCAATCCGGACCAA /5Phos/GTTGTTGGTCCGGATTGGTCCGGA TCCGTAACGGTCCGTAACGG /5Phos/ CCCTCCGTAACGGTCCGTAACGG /5Phos/GTTGCCGTTACGGACCGTTACGGA TCCGTAGGTCTCCGTAGGTC /5Phos/ CCCTCCGTAGGTC CCGTAGGTC /5Phos/GTTGGACCTACGGAGACCTACGGA TCCGTCCAAGTCCGTCCAAG /5Phos/ CCCTCCGTCCAAGTCCGTCCAAG /5Phos/GTTGCTTGGACGGACTTGGACGGA TCCTGAACCGTCCTGAACCG /5Phos/TCCCTCCTGAACCGTCCTGAACCG /5Phos/GTTGCGGTTCAGGACGGTTCAGGA TCCTGGCATGTCCTGGCATG /5Phos/TCCCTCCTGGCATGTCCTGGCATG /5Phos/GTTGCATGCCAGGACATGCCAGGA TCGCGCTACATCGCGCTACA /5Phos/TCCCTCGCGCTACATCGCGCTACA /5Phos/GTTGTGTAGCGCGATGTAGCGCGA TCGGTTACCATCGGTTACCA /5Phos/ CCCTCGGTTACCATCGGTTACCA /5Phos/GTTGTGGTAACCGATGGTAACCGA TCGTCCGTCATCGTCCGTCA /5Phos/ CCCTCGTCCGTCATCGTCCGTCA /5Phos/GTTGTGACGGACGATGACGGACGA TCGTCCTCAGTCGTCCTCAG /5Phos/ CCCTCGTCCTCAGTCGTCCTCAG /5Phos/GTTGCTGAGGACGACTGAGGACGA TCTACCGCTCTCTACCGCTC /5Phos/TCCCTCTACCGCTCTCTACCGCTC /5Phos/GTTGGAGCGGTAGAGAGCGGTAGA TCTAGCTCGGTCTAGCTCGG /5Phos/ CCCTCTAGCTCGGTCTAGCTCGG /5Phos/GTTGCCGAGCTAGACCGAGCTAGA TCTCGGCTGATCTCGGCTGA /5Phos/TCCCTCTCGGCTGATCTCGGCTGA /5Phos/GTTGTCAGCCGAGATCAGCCGAGA TCTCGGTCAGTCTCGGTCAG /5Phos/TCCCTCTCGGTCAGTCTCGGTCAG /5Phos/GTTGCTGACCGAGACTGACCGAGA TCTCTAGATCTCTCTAGATC /5Phos/TCCCTCTCTAGATCTCTCTAGATC /5Phos/GTTGGATCTAGAGAGATCTAGAGA TCTGACCGCATCTGACCGCA /5Phos/TCCCTCTGACCGCATCTGACCGCA /5Phos/GTTGTGCGGTCAGATGCGGTCAGA TCTGGACAGATCTGGACAGA /5Phos/TCCCTCTGGACAGATCTGGACAGA /5Phos/GTTGTC GTCCAGATCTGTCCAGA TCTGGATAAGTCTGGATAAG /5Phos/TCCCTCTGGATAAGTCTGGATAAG /5Phos/GTTGCTTATCCAGACTTATCCAGA TCTTACGGCCTCTTACGGCC /5Phos/TCCCTCTTACGGCCTCTTACGGCC /5Phos/GTTGGGCCGTAAGAGGCCGTAAGA TCTTGCATACTCTTGCATAC /5Phos/ CCCTCTTGCATACTCTTGCATAC /5Phos/GTTGGTATGCAAGAGTATGCAAGA TGAACTTGGATGAACTTGGA /5Phos/ CCCTGAACTTGGATGAACTTGGA /5Phos/GTTGTCCAAGTTCATCCAAGTTCA TGACACAGCGTGACACAGCG /5Phos/ CCCTGACACAGCGTGACACAGCG /5Phos/GTTGCGCTGTGTCACGCTGTGTCA TGACAGGTCCTGACAGGTCC /5Phos/ CCCTGACAGGTCCTGACAGGTCC /5Phos/GTTGGGACCTGTCAGGACCTGTCA TGACCTTCCGTGACCTTCCG /5Phos/TCCCTGACCTTCCGTGACCTTCCG /5Phos/GTTGCGGAAGGTCACGGAAGGTCA TGACGTCGGATGACGTCGGA /5Phos/TCCCTGACGTCGGATGACGTCGGA /5Phos/GTTGTCCGACGTCATCCGACGTCA TGACTATCTCTGACTATCTC /5Phos/ CCCTGACTATCTCTGACTATCTC /5Phos/GTTGGAGATAGTCAGAGATAGTCA TGACTCCAGGTGACTCCAGG /5Phos/ CCCTGACTCCAGGTGACTCCAGG /5Phos/GTTGCCTGGAGTCACCTGGAGTCA TGAGAGCAGGTGAGAGCAGG /5Phos/ CCCTGAGAGCAGGTGAGAGCAGG /5Phos/GTTGCCTGCTCTCACCTGCTCTCA TGAGCCTCCATGAGCCTCCA /5Phos/ CCCTGAGCCTCCATGAGCCTCCA /5Phos/GTTGTGGAGGCTCATGGAGGCTCA TGAGTATGGATGAGTATGGA /5Phos/ CCCTGAGTATGGATGAGTATGGA /5Phos/GTTGTCCATACTCATCCATACTCA TGCAACATACTGCAACATAC /5Phos/ CCCTGCAACATACTGCAACATAC /5Phos/GTTGGTATGTTGCAGTATGTTGCA TGCGCTGTAGTGCGCTGTAG /5Phos/TCCCTGCGCTGTAGTGCGCTGTAG /5Phos/GTTGCTACAGCGCACTACAGCGCA TGCTAACCGGTGCTAACCGG /5Phos/TCCCTGCTAACCGGTGCTAACCGG /5Phos/GTTGCCGGTTAGCACCGGTTAGCA TGCTCCACTGTGCTCCACTG /5Phos/TCCCTGCTCCACTGTGCTCCACTG /5Phos/GTTGCAGTGGAGCACAGTGGAGCA TGGAAGGAGCTGGAAGGAGC /5Phos/TCCCTGGAAGGAGCTGGAAGGAGC /5Phos/GTTGGCTCCTTCCAGCTCCTTCCA TGGCAGTGACTGGCAGTGAC /5Phos/ CCCTGGCAGTGACTGGCAGTGAC /5Phos/GTTGGTCACTGCCAGTCACTGCCA TGGCATCAGCTGGCATCAGC /5Phos/TCCCTGGCATCAGCTGGCATCAGC /5Phos/GTTGGCTGATGCCAGCTGATGCCA TGGCCTTATATGGCCTTATA /5Phos/TCCCTGGCCTTATATGGCCTTATA /5Phos/GTTGTATAAGGCCATATAAGGCCA TGGCGAAGCATGGCGAAGCA /5Phos/ CCCTGGCGAAGCATGGCGAAGCA /5Phos/GTTGTGCTTCGCCATGCTTCGCCA TGGCTTAAGATGGCTTAAGA /5Phos/TCCCTGGCTTAAGATGGCTTAAGA /5Phos/GTTGTCTTAAGCCATCTTAAGCCA TGGTCAGCTCTGGTCAGCTC /5Phos/TCCCTGGTCAGCTCTGGTCAGCTC /5Phos/GTTGGAGC GACCAGAGCTGACCA TGGTTCCAACTGGTTCCAAC /5Phos/ CCCTGGTTCCAACTGGTTCCAAC /5Phos/GTTGGTTGGAACCAGTTGGAACCA TGGTTGGTAATGGTTGGTAA /5Phos/ CCCTGGTTGGTAATGGTTGGTAA /5Phos/GTTGTTACCAACCATTACCAACCA TGTACGCGCATGTACGCGCA /5Phos/TCCCTGTACGCGCATGTACGCGCA /5Phos/GTTGTGCGCGTACATGCGCGTACA TGTACGCTGGTGTACGCTGG /5Phos/ CCCTGTACGCTGGTGTACGCTGG /5Phos/GTTGCCAGCGTACACCAGCGTACA TGTAGCATTG GTAGCAT G /5Phos/TCCCTGTAGCAT GTGTAGCATTG /5Phos/GTTGCAATGCTACACAATGCTACA TGTAGTGCCGTGTAGTGCCG /5Phos/TCCCTGTAGTGCCGTGTAGTGCCG /5Phos/GTTGCGGCACTACACGGCACTACA TGTGAATCTGTGTGAATCTG /5Phos/TCCCTGTGAATCTGTGTGAATCTG /5Phos/GTTGCAGATTCACACAGATTCACA TGTTCCGTGGTGTTCCGTGG /5Phos/TCCCTGTTCCGTGGTGTTCCGTGG /5Phos/GTTGCCACGGAACACCACGGAACA TTAGCGCGTGTTAGCGCGTG /5Phos/ CCCTTAGCGCGTGTTAGCGCGTG /5Phos/GTTGCACGCGCTAACACGCGCTAA TTAGTTGGACTTAGTTGGAC /5Phos/ CCCTTAGTTGGACTTAGTTGGAC /5Phos/GTTGGTCCAACTAAGTCCAACTAA TTCCAACTTCTTCCAACTTC /5Phos/TCCCTTCCAACTTCTTCCAACTTC /5Phos/GTTGGAAGTTGGAAGAAGTTGGAA TTCCGTCAGGTTCCGTCAGG /5Phos/TCCCTTCCGTCAGGTTCCGTCAGG /5Phos/GTTGCCTGACGGAACCTGACGGAA TTCCTCACCGTTCCTCACCG /5Phos/TCCCTTCCTCACCGTTCCTCACCG /5Phos/GTTGCGGTGAGGAACGGTGAGGAA TTCCTCCGACTTCCTCCGAC /5Phos/TCCCTTCCTCCGACTTCCTCCGAC /5Phos/GTTGGTCGGAGGAAGTCGGAGGAA TTCGACCTGGTTCGACCTGG /5Phos/TCCCTTCGACCTGGTTCGACCTGG /5Phos/GTTGCCAGGTCGAACCAGGTCGAA TTCGCGTGGATTCGCGTGGA /5Phos/TCCCTTCGCGTGGATTCGCGTGGA /5Phos/GTTGTCCACGCGAATCCACGCGAA TTCGGAAGCGTTCGGAAGCG /5Phos/ CCCTTCGGAAGCGTTCGGAAGCG /5Phos/GTTGCGCTTCCGAACGCTTCCGAA TTCTCGATTGTTCTCGATTG /5Phos/ CCCTTCTCGATTGTTCTCGATTG /5Phos/GTTGCAATCGAGAACAATCGAGAA TTCTCTCTAGTTCTCTCTAG /5Phos/TCCCTTCTCTCTAGTTCTCTCTAG /5Phos/GTTGCTAGAGAGAACTAGAGAGAA TTCTTGCGCGTTCTTGCGCG /5Phos/ CCCTTCTTGCGCGTTCTTGCGCG /5Phos/GTTGCGCGCAAGAACGCGCAAGAA TTGAGGTCGGTTGAGGTCGG /5Phos/ CCCTTGAGGTCGGTTGAGGTCGG /5Phos/GTTGCCGACCTCAACCGACCTCAA TTGCACACGCTTGCACACGC /5Phos/TCCCTTGCACACGCTTGCACACGC /5Phos/GTTGGCGTGTGCAAGCGTGTGCAA TTGCGCTCTGTTGCGCTCTG /5Phos/ CCCTTGCGCTCTGTTGCGCTCTG /5Phos/GTTGCAGAGCGCAACAGAGCGCAA TTGCTGCAGCTTGCTGCAGC /5Phos/ CCCTTGCTGCAGCTTGCTGCAGC /5Phos/GTTGGCTGCAGCAAGCTGCAGCAA TTGGCGTCTGTTGGCGTCTG /5Phos/ CCCTTGGCGTCTGTTGGCGTCTG /5Phos/GTTGCAGACGCCAACAGACGCCAA TTGGTCCTTCTTGGTCCTTC /5Phos/TCCCTTGGTCCTTCTTGGTCCTTC /5Phos/GTTGGAAGGACCAAGAAGGACCAA TTGTCGAGAGTTGTCGAGAG /5Phos/TCCCTTGTCGAGAGTTGTCGAGAG /5Phos/GTTGCTCTCGACAACTCTCGACAA TTGTCTGTACTTGTCTGTAC /5Phos/ CCCTTGTCTGTACTTGTCTGTAC /5Phos/GTTGGTACAGACAAGTACAGACAA TTGTTCTCTCTTGTTCTCTC /5Phos/ CCCTTGTTCTCTCTTGTTCTCTC /5Phos/GTTGGAGAGAACAAGAGAGAACAA AACACACGTTAACACACGTT /5Phos/ CCCAACACACGTTAACACACGTT /5Phos/GTTGAACGTGTGTTAACGTGTGTT AACATCGAGTAACATCGAGT /5Phos/ CCCAACATCGAGTAACATCGAGT /5Phos/GTTGACTCGATGTTACTCGATGTT AACCGAGGACAACCGAGGAC /5Phos/ CCCAACCGAGGACAACCGAGGAC /5Phos/GTTGGTCCTCGGTTGTCCTCGGTT AACCTAGTAGAACCTAGTAG /5Phos/ CCCAACCTAGTAGAACCTAGTAG /5Phos/GTTGCTACTAGGTTCTACTAGGTT AACGCATACTAACGCATACT /5Phos/ CCCAACGCATACTAACGCATACT /5Phos/GTTGAGTATGCGTTAGTATGCGTT AACTAACCTCAACTAACCTC /5Phos/TCCCAACTAACCTCAACTAACCTC /5Phos/GTTGGAGGTTAGTTGAGGTTAGTT AACTAAGGAGAACTAAGGAG /5Phos/TCCCAACTAAGGAGAACTAAGGAG /5Phos/GTTGCTCCTTAGTTCTCCTTAGTT AACTCGTTCTAACTCGTTCT /5Phos/ CCCAACTCGTTCTAACTCGTTCT /5Phos/GTTGAGAACGAGTTAGAACGAGTT AACTGCCGCTAACTGCCGCT /5Phos/TCCCAACTGCCGCTAACTGCCGCT /5Phos/GTTGAGCGGCAGTTAGCGGCAGTT
[0109] Each unique label comprises two or more detectable oligonucleotide tags. The two or more tags may be three or more tags, four or more tags, or five or more tags. In some embodiments, a unique label comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more detectable tags.
[0110] The tags are typically bound to each other, typically in a directional manner.
Methods for sequentially attaching nucleic acids such as oligonucleotides to each other are known in the art and include, but are not limited to, ligation and polymerization, or a combination of both (see, e.g., Green and Sambrook. Molecular Cloning: A Laboratory Manual, Fourth Edition, 2012). Ligation reactions include blunt end ligation and cohesive overhang ligation. In some instances, ligation may comprise both blunt end and cohesive overhang ligation. A cohesive overhang is a single stranded end sequence (attached to a double stranded sequence) capable of binding to another single stranded sequence thereby forming a double stranded sequence. A cohesive overhang may be generated by a polymerase, a restriction endonuclease, a combination of a polymerase and a restriction endonuclease, or a Uracil-Specific Excision Reagent (USER™) enzyme (New England BioLabs Inc., Ipswich, MA) or a combination of a Uracil DNA glycosylase enzyme and a DNA glycosylase-lyase Exonuclease VIII enzyme. A cohesive overhang may be a thymidine tail. Polymerization reactions include enzyme-mediated polymerization such as a polymerase-mediated fill-reaction.
[0111] Methods for detecting and analyzing unique labels are known in the art. In some embodiments, detection comprises determining the presence, number, and/or order of detectable tags that comprise a unique label. Methods of sequencing oligonucleotides and nucleic acids are well known in the art (see, e.g., W093/23564, WO98/28440 and W098/13523; U.S. Pat. Nos. 5,525,464; 5,202,231; 5,695,940; 4,971,903; 5,902,723; 5,795,782; 5,547,839 and 5,403,708; Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977); Drmanac et al., Genomics 4:114 (1989); Koster et al., Nature Biotechnology 14:1123 (1996); Hyman, Anal. Biochem. 174:423 (1988); Rosenthal, International Patent Application Publication 761107 (1989); Metzker et al., Nucl. Acids Res. 22:4259 (1994); Jones, Biotechniques 22:938 (1997); Ronaghi et al., Anal. Biochem. 242:84 (1996); Ronaghi et al., Science 281:363 (1998); Nyren et al., Anal. Biochem. 151 :504 (1985); Canard and Arzumanov, Gene 11:1 (1994); Dyatkina and Arzumanov, Nucleic Acids Symp Ser 18:117 (1987); Johnson et al., Anal. Biochem.136: 192 (1984); and Elgen and Rigler, Proc. Natl. Acad. Sci. USA 91(13):5740 (1994), all of which are expressly incorporated by reference).
Generation of Unique Labels
[0112] In some aspects, the invention provides methods for generating unique labels. The methods typically use a plurality of detectable tags to generate unique labels. In some embodiments, a unique label is produced by sequentially attaching two or more detectable oligonucleotide tags to each other. The detectable tags may be present or provided in a plurality of detectable tags. The same or a different plurality of tags may be used as the source of each detectable tag comprised in a unique label. In other words, a plurality of tags may be subdivided into subsets and single subsets may be used as the source for each tag. This is exemplified in at least FIG. 1.
[0113] A plurality of tags may comprise 2, 3 , 4, 5, 6, 7, 8, 9, 10, 102, 103, 104, 105, or 106, or more tags. Typically, the tags within a plurality are unique relative to each other.
[0114] The methods of the invention allow an end user to generate a unique label for a plurality of agents using a number of tags that are less (and in some instances far less) than the number of agents to be labeled. The number of tags may be up to or about 10-fold, 10 -fold, 10 - fold, or 104-fold less than the number of agents. The number of agents to be labeled will depend on the particular application. The invention contemplates uniquely labeling at least 103, 104, 105, 10°, 10', 10°, 10% or 10ιυ or more agents. In some embodiments, the agent comprises a plurality of nucleic acids. In some embodiments, the plurality of nucleic acids comprises at least 103, 104, 105, 106, 107, 10s, 109, or 1010 nucleic acids.
[0115] In certain methods of the invention, agents, detectable tags, and resultant unique labels are all present in a contained volume and are thus physically separate from other agents, detectable tags, and resultant unique labels. In some instances, the contained volume is on the order of picoliters, nanoliters, or microliters. The contained volume may be a droplet such as an emulsion droplet.
[0116] As discussed herein, in some instances, an agent is attached to the unique label (or label intermediate) directly or indirectly. In these instances, once the detectable tag is attached to either the agent or the label intermediate, the droplets are ruptured (or broken) and their contents are pooled (and effectively mixed together). The contents of the pool may be introduced, at limiting dilution, into another plurality of emulsion droplets each of which comprises a single detectable oligonucleotide tag (and optionally multiple copies of the oligonucleotide tag). This results in droplets that each contain an agent or a label intermediate, together with a single detectable oligonucleotide tag, and optionally reagents and enzymes required for tag attachment. Once the tag is attached, the droplets are again ruptured, and the process is repeated until a sufficient number of unique labels is generated. [0117] In some embodiments a subset of the plurality of agents is present in the same container during attachment of a detectable label. In some embodiments, the plurality of agents is separated such that each agent in the plurality is in a separate container, e.g., an emulsion droplet.
[0118] In some embodiments, the process of pooling and subsequently separating the plurality of agents is performed n number of times, wherein n is the number of times required to generate (mi)(m2)(m3)...(mn) number of combinations of detectable oligonucleotide tags, wherein (m1)(m2)(m3)...(mn) number of combinations of detectable oligonucleotide tags is greater than the number of the plurality of agents.
[0119] The invention provides a method comprising
(a) labeling two or more first subsets of agents with a detectable oligonucleotide tag to produce agents within a subset that are identically labeled relative to each other and uniquely labeled relative to agents in other subsets;
(b) combining two or more subsets of uniquely-labeled agents to form a pool of agents, wherein the pool comprises two or more second subsets of agents that are distinct from the two or more first subsets of agents;
(c) identically labeling two or more second subsets of agents with a second detectable oligonucleotide tag to produce agents within a second subset that are uniquely labeled relative to agents in the same or different second subsets; and
(d) repeating steps (b) and (c) until a number of unique labels is generated that exceeds the number of starting agents, wherein each unique label comprises at least two detectable oligonucleotide tags.
[0120] The invention provides another method comprising
(a) providing a pool of agents;
(b) separating the pool of agents into sub-pools of agents;
(c) labeling agents in each sub-pool of with one of in] unique detectable oligonucleotide tags thereby producing sub-pools of labeled agents, wherein agents in a sub-pool are identically labeled to each other;
(d) combining sub-pools of labeled agents to create a pool of labeled agents;
(e) separating the pool of labeled agents into second sub-pools of agents; repeating steps (c) to (e) n times to produce agents labeled with n unique detectable oligonucleotide tags, wherein the pool in (a) consists of a number of agents that is less than (mi)(m2)(ni3) ... (mn).
[0121] The invention provides another method comprising
providing a population of library droplets comprising agents, wherein each droplet comprises an agent;
fusing each individual library droplet with a single detectable oligonucleotide tag droplet from a plurality of ni! detectable oligonucleotide tag droplets, each detectable oligonucleotide tag droplet comprising a plurality of identical detectable oligonucleotide tag;
labeling the agent with the detectable oligonucleotide tag in a fused droplet; harvesting labeled agents from the fused droplets and generating another population of library droplets comprising labeled agents; and repeating steps (b) to (d) n times to produce agents labeled with a unique label comprising n detectable oligonucleotides tags, wherein the n detectable oligonucleotide tags generate an (mi)(m2)(m3)...(mn) number of combinations that is greater than the number of starting agents.
[0122] The invention provides another method comprising
providing a population of library droplets comprising agents, wherein each droplet comprises more than one agent;
fusing each individual library droplet with a single detectable oligonucleotide tag droplet from a plurality of m detectable oligonucleotide tag droplets, each detectable oligonucleotide tag droplet comprising a plurality of identical detectable oligonucleotide tag;
labeling the more than one agents with the detectable oligonucleotide tag in a fused droplet;
harvesting labeled agents from the fused droplets and generating another population of library droplets comprising labeled agents, wherein each droplet comprises more than one agent; and
repeating steps (b) to (d) n times to produce agents labeled with a unique label comprising n detectable oligonucleotides tags, wherein the n detectable oligonucleotide tags generate an (mj)(m2)(m3)...(mn) number of combinations that is greater than the number of starting agents and optionally wherein the agents within the same droplet are labeled identically.
Emulsions
[0123] Methods for making emulsion droplets are known in the art (see, e.g., WO 2006/040551 ; WO 2006/040554; WO 2004/002627; WO 2004/091763; WO 2005/021151; WO 2006/096571; WO 2007/089541 ; WO 2007/081385 and WO 2008/063227; U.S. Patent No. 7,708,949; U.S. Patent Publication No. 20120122714, 20110000560, 20100022414; John Leamon, Darren R Link, Michael Egholm & Jonathan M Rothberg. Overview: methods and applications for droplet compartmentalization of biology. Nature Methods. Vol. 3, No. 7, 2006; E. Brouzes, M. Medkova, N. Savenelli, D. Marran, M. Twardowski, J.B. Hutchison, J.M. Rothberg, D.R. Link, N. Perrimon, M.L. Samuels. Droplet microfluidic technology for single- cell high-throughput screening. PNAS, 106, 14195. 2009; J.C. Baret, O.J. Miller, V. Taly, M. Ryckelynck, A. El-Harrak, L. Frenz, C. Rick, M.L. Samuels, J.B. Hutchison, J.J. Agresti, D.R. Link, D.A. Weitz and A.D. Griffiths. Fluorescence-activated droplet sorting (FADS): Efficient microfluidic cell sorting based on enzymatic activity. Lab Chip, 9, 1850. 2009; M.M. Kiss, L. Ortoleva-Donnelly, N.R. Beer, J. Warner, C.G. Bailey, B.W. Colston, J.M. Rothberg, D.R. Link, and J.H. Leamon. High-throughput quantitative polymerase chain reaction in pico liter droplets. Anal Chem. 2008 December 1 ; 80(23): 8975-8981 ; Edd et al. Controlled encapsulation of single-cells into monodisperse picolitre drops. Lab Chip, 8(8): 1262-1264, 2008; Anna SL, Bontoux N, Stone HA (2003) Formation of dispersions using "flow focusing" in microchannels. Appl Phys Lett 82:364-366; all of which are incorporated herein by reference in their entirety).
[0124] A "droplet" or "emulsion droplet", as used herein, is an isolated portion of a first fluid that is completely surrounded by a second fluid. The first and second fluids are immiscible with each other. For example, the discontinuous phase can be an aqueous solution and the continuous phase can a hydrophobic fluid such as an oil or a fluorocarbon oil. This is termed a water in oil emulsion. Alternatively, the emulsion may be an oil in water emulsion. In that example, the first liquid, which is dispersed in globules, is referred to as the discontinuous phase, whereas the second liquid is referred to as the continuous phase or the dispersion medium. The continuous phase can be an aqueous solution and the discontinuous phase is a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, or hexadecane). The droplets or globules of oil in an oil in water emulsion are also referred to herein as "micelles", whereas globules of water in a water in oil emulsion may be referred to as "reverse micelles". In some cases, the droplets may be spherical or substantially spherical; however, in other cases, the droplets may be non-spherical.
[0125] The terms "droplet library" or "droplet libraries" are also referred to herein as an "emulsion library" or "emulsion libraries." Examples of droplet libraries are collections of droplets that have different contents, ranging from DNA, primers, etc. The droplets range in size from roughly 0.5 micron to 500 micron in diameter, which corresponds to about 1 pico liter to 1 nano liter. However, droplets can be as small as 5 microns and as large as 500 microns. Preferably, the droplets are at less than 100 microns, about 1 micron to about 100 microns in diameter. The most preferred size is about 20 to 40 microns in diameter (10 to 100 picoliters). The preferred properties examined of droplet libraries include osmotic pressure balance, uniform size, and size ranges.
Droplets can be generated by infusing aqueous samples comprising library elements, e.g., agents, detectable tags, or combinations thereof, at a perpendicular angle to opposing oil streams. Droplets can be contained within a microfluidic channel. Microfluidic channels and method for manufacturing microfluidic channels are known in the art (see, e.g., McDonald JC, et al. (2000) Fabrication of microfluidic systems in poly(dimethylsiloxane) Electrophoresis 21:27-40; Siegel AC, et al.
[0126] (2006) Cofabrication of electromagnets and microfluidic systems in poly(dimethylsiloxane)13. Angew Chem-Ger Edit 118:7031-7036; Agresti et al. (2009). Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. PNAS 107(9), 4004-4009; all of which are incorporated herein in their entirety).
[0127] Other examples of microfluidic devices and approaches for use herein are disclosed in an application filed on September 21, 2012, entitled "Systems and Methods for Droplet Tagging", incorporated by reference herein in its entirety.
[0128] Droplets can be optionally merged. Merging can be accomplished, e.g., by passing an electrical field through a microfluidic channel to merge charged droplets, or by addition of a chemical that breaks emulsions. (See K. Ahn, J. Agresti, H. Chong, M. Marquez and D. A. Weitz, Appl. Phys. Lett., 2006, 88, 264105 and D Link, E Grasland-Mongrain, A Duri, F Sarrazin, Z Cheng, G Cristobal, M Marquez, and DA Weitz. Angew. Chem. Int. Ed. 2006, 45, 2556 -2560 as examples.)
[0129] Generation of unique labels may occur in part or entirely in emulsion droplets. The unique label is generated in an emulsion droplet or in a series of emulsion droplets. A library of uniquely-labeled agents is generated using an emulsion droplet or a series of emulsion droplets.
Mate-Pair Analysis
[0130] Another aspect of the invention addresses a fundamental issue associated with the creation of mate-pair (long distance linkage) libraries. Mate-pair libraries are useful for extracting distance information from sequences and are most typically used in genomic assemblies, detection of splicing in transcripts, and detection of genomic rearrangements. Traditionally, mate-pair libraries require that DNA molecules be circularized in order to directly join the ends together (i.e., as a mate-pair). The efficiency of circularization decreases as jump length increases, thus increasingly specialized techniques are required in order to prepare jumps of varying sizes. The methods described herein offer a major advantage over current methodologies in that mate-pair analysis is achieved without relying on circularization and is independent of jump length, thus making it a universal mate-pair protocol potentially suitable across a range of sequencing technologies.
[0131] In some embodiments, reactions are performed in emulsion droplets at single molecule dilution resulting in significant reductions in reagent costs, cycle time and input material. As described herein, emulsion droplets are used to segregate individual DNA molecules so that the ends of each DNA molecule can either be physically re-joined via ligation or informatically associated via analysis of the unique label.
[0132] Accordingly, in one aspect methods are provided for performing mate-pair analysis.
[0133] In some embodiments, the method comprises:
(a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acids end-labeled on its 5' and 3' ends with oligonucleotide label, wherein the oligonucleotide label on the 5' end (the 5' oligonucleotide label) and the oligonucleotide on the 3' end (the 3' oligonucleotide label) comprise a nucleotide cohesive overhang, and wherein the nucleotide cohesive overhang on the 5' oligonucleotide label is complementary to the nucleotide cohesive overhang on the 3 ' oligonucleotide label;
(b) fusing each individual library droplet with a droplet comprising a DNA fragmenting enzyme, thereby producing a fused droplet;
(c) fragmenting the nucleic acid with the 5' and 3' oligonucleotide labels in the fused droplet, thereby producing a fused droplet comprising a nucleic acid fragment comprising the 5' oligonucleotide label and a nucleic acid fragment comprising the 3' oligonucleotide label; and
(d) ligating the 5' oligonucleotide label and the 3' oligonucleotide label nucleic acid, thereby ligating the nucleic acid fragment comprising the 5' oligonucleotide label and the nucleic acid fragment comprising the 3' oligonucleotide label, thereby producing a ligated nucleic acid.
[0134] In some embodiments, the 5' oligonucleotide label and/or the 3' oligonucleotide comprises a biotin label. In some embodiments, the method further comprises (e) sequencing the ligated nucleic acid. In some embodiments, the DNA fragmenting agent is Nextera.
[0135] In another embodiment, the method comprises:
(a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acid comprising an oligonucleotide adapter;
(b) melting the nucleic acid;
(c) fusing each individual library droplet comprising a melted nucleic acid with a single index droplet from a plurality of m index droplets, each index droplet comprising a first unique single-stranded detectable oligonucleotide tag, wherein the first unique single-stranded detectable oligonucleotide tag comprises a region complementary to the oligonucleotide adapter;
(d) annealing the first unique single-stranded detectable oligonucleotide tag to the nucleic acid and performing a fill-in reaction, thereby producing an end- labeled nucleic;
(e) harvesting end-labeled nucleic acids from the fused droplets and generating another population of library droplets, wherein each droplet comprises an end- labeled nucleic acid;
(f) melting the end-labeled nucleic acid; (g) fusing each individual library droplet comprising a melted end-labeled nucleic acid with a single index droplet from a plurality of m2 index droplets, each index droplet comprising a second unique single-stranded detectable oligonucleotide tag, wherein the second unique single-stranded detectable oligonucleotide tag comprises a region complementary to the first unique single-stranded detectable oligonucleotide tag;
(h) annealing the second unique single-stranded detectable oligonucleotide tag to the nucleic acid and performing a fill-in reaction, thereby producing an end- labeled nucleic acid;
(i) harvesting end-labeled nucleic acid molecules from the fused droplets and generating another population of library droplets comprising end-labeled nucleic acids; and
(j) repeating steps (f) to (i) n times to produce nucleic acids end-labeled with n detectable oligonucleotide tags, wherein the n detectable oligonucleotide tags generate an (mi)(m2)(m3)...(mn) number of combinations that is greater than the number of starting nucleic acids.
[0136] In another embodiment, the method comprises:
sequentially end-labeling nucleic acids in a plurality, at their 5' and 3' ends, with a random combination of n detectable oligonucleotide tags, wherein each end-labeled nucleic acid is
(a) identically labeled at its 5' and 3' ends, and
(b) uniquely labeled relative to other nucleic acids in the plurality, wherein each detectable oligonucleotide tags is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of a nucleic acid.
[0137] In some embodiments, the further comprises fragmenting end-labeled nucleic acids into at least a 5' fragment comprising the 5' end of the nucleic acid attached to the random combination of n detectable oligonucleotide tags and into a 3' fragment comprising the 3' end of the nucleic acid attached to the random combination of n detectable oligonucleotide tags. In some embodiments, the 5' and 3' fragments are about 10-1000 bases (base pairs) in length, or about 10-500 bases in length, or about 10-200 bases in length. In some embodiments, the method further comprises sequencing the 5' and 3' fragments.
[0138] In another embodiment, the method comprises:
sequencing a pair of genomic nucleic acid fragments, wherein the genomic nucleic acid fragments are attached to identical unique labels at one of their ends that indicates the genomic nucleic acid fragments were separated by a known distance in a genome prior to fragmentation.
[0139] In some embodiments, the pair of nucleic acid fragments were separated by greater than 10 kb in the genome prior to fragmentation. In another embodiment, the pair of nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation. In some embodiments, the method further comprises generating the pair of genomic nucleic acid fragments by fragmenting nucleic acids comprising genomic sequence and identical non- genomic sequence at their 5' and 3' ends.
[0140] In another aspect, the invention provides compositions. In one embodiment, the composition comprises:
a plurality of paired nucleic acid fragments attached to unique labels at one end, wherein paired nucleic acid fragments:
(a) share an identical unique label at one end that is unique in the plurality, and
(b) were separated from each other in a genome by a known distance prior to fragmentation.
[0141] In some embodiments, paired nucleic acid fragments were separated by greater than 10 kb in the genome prior to fragmentation. In another embodiment, the paired nucleic acid fragments were separated by greater than 40 kb in the genome prior to fragmentation. In some embodiments, the composition is produced using any of the methods described herein.
[0142] Examples of nucleic acids include, but are not limited to, genomic DNA, cDNA, PCR products, mRNA, total RNA, plasmids, or fragments thereof. In some embodiments, the nucleic acids are genomic DNA, cDNA, PCR products, or fragments thereof. Nucleic acids can be fragmented using methods described herein. [0143] In some embodiments, the method further comprises fragmenting uniquely end- labeled nucleic acids. Fragmenting of nucleic acids can be accomplished by methods described herein and those well-known in the art.
[0144] In one embodiment, the method comprises sequencing a pair of genomic nucleic acid fragments, wherein the genomic nucleic acid fragments are attached to identical unique labels at one of their ends that indicates the genomic nucleic acid fragments were separated by a known distance in a genome prior to fragmentation. In some embodiments, the known distance is greater than 5, 10, 15, 20, 30, 40, 50, 100 kb or greater separation. Genomic nucleic acid fragments can come from any organismal genomic DNA, for example, human, mammalian, bacterial, fungal or plant genomic DNA. Genomic nucleic acid fragments can be generated by fragmentation methods known in the art (see, e.g., Green and Sambrook. Molecular Cloning: A Laboratory Manual, Fourth Edition, 2012). Examples of fragmentation include, but are not limited to, enzymatic (such as a nuclease), chemical (such as a DNA nicking agent) or mechanical (such as sonication) fragmentation. Fragmentation can be random, e.g., sequence and size unspecific, or ordered, e.g., sequence dependent and/or size-restricted. The fragments generated following label addition can be tailored to the limitations of the desired detection technology. For example, the fragments can be hundreds, thousands, millions or potentially billions of base pairs in length depending on the technology used to sequence the DNA.
EXAMPLES
[0145] The following Examples are meant for illustrative purposes, and are not meant to be exclusive or limiting.
Example 1: Polymer -ase-mediated bioinformatic association of nucleic acid ends for Mate-Pair Analysis
Generation of end-labeled genomic DNA fragments
[0146] A method for bioinformatically associating the ends of genomic DNA is outlined in Figure 2. Genomic DNA is fragmented and size selected to a known size using techniques known in the art (e.g., sonication, cavitation, point-sink or mechanical shearing, or a DNA fragmenting enzyme and size-exclusion columns or gel purification). The genomic DNA is then A-tailed and ligated to a biotinylated, T-tailed asymmetric oligonucleotide adapter using methods known in the art (see, e.g. Maniatis, Molecular Cloning). Klenow exo- enzyme is commonly used to add a single nucleotide to the 3' termini of DNA fragments). The adapter is a partial duplex to allow for annealing of the single-stranded oligonucleotide indexes described below.
[0147] One or more index libraries (preferably 1-4 libraries) are created such that each library contains approximately >1000 unique single-stranded oligonucleotide indexes, thus approximately 2000-4000 unique indexes are used. Index libraries may be created in droplets using standard flow focusing techniques. For a given library, each droplet will contain many copies of one unique single-stranded index. Droplets may contain some or all of the key components of a polymerase fill-in reaction (e.g., MgCl2, dNTP, and Polymerase). Each unique single-stranded oligonucleotide index contains 3 distinct regions: sequence complimentary to the adapter (Ad) or to a previously added index sequence (B or C), a unique index sequence (Idx), and a sequence used to "capture" the next index oligonucleotide index which contains one or more dUTP nucleotides (B7C).
[0148] Fragmented genomic DNA ligated to an adapter is diluted to a desired concentration to control the number of molecules per droplet (e.g., a single DNA molecule per droplet or more than a single DNA molecule per droplet) and merged with (see above references for droplet merging) the first index library (Library "A" in Figure 2).
[0149] The Ad region of each unique single-stranded oligonucleotide index binds to the adapter on each end of the fragmented genomic DNA molecule. A polymerase-mediated fill-in reaction is performed in each droplet, creating the complement to the index and capture regions on the each unique single-stranded oligonucleotide index a, and thus generating unique double- stranded oligonucleotide indexes.
[0150] Emulsion droplets are then broken using various mechanical or chemical reagents depending on the oil/surfactant utilized in the emulsion, resulting in the combination and mixing of the DNA from each droplet. Mixed DNA is then treated with USER™ enzyme (Uracil- Specific Excision Reagent, New England BioLabs Inc., Ipswich, MA), causing the capture portion of the double-stranded oligonucleotide index to be digested due to the presence of one or more dUTP nucleotides. This digestion reveals the nascent strand, which is complementary to a sequence contained in the next library of indexes (Library "B" in Figure 2).
[0151] The process of fragmented genomic DNA dilution, merging with a droplet library, polymerase fill-in, breaking the droplets, and treatment with USER™ enzyme is repeated for the desired number of cycles, each time adding one new unique oligonucleotide index sequence to both ends of the fragmented genomic DNA.
[0152] After the final index addition, the result is fragmented genomic DNA uniquely end- labeled on both the 5' and 3' end with a unique label made up of many oligonucleotide indexes. The uniquely end-labeled fragmented genomic is then fragmented and the ends are collected via streptavidin beads, which recognized the biotin label on the adapter. Fragments can be ligated to technology specific sequencing adapters (e.g., Illumina adapters) and sequenced. Ends are informatically paired by matching the unique label on one fragment of DNA with the same unique label on the other fragment of DNA (see Figure 7).
[0153] This method of bioinformatics association can also be used with other types of nucleic acids, such as RNA, cDNA, or PCR-amplified DNA, or any other type of construct where such a labeling scheme is required.
Example 2: Ligation-mediated bioinformatic association of nucleic acid ends in emulsions for Mate-Pair Analysis
Validation of Ligation in Emulsions
[0154] A 34 bp adapter was designed. The adapter was biotinylated and T-tailed to force directionality of ligation to A-tailed lambda genomic DNA. Ligation was performed in an tube or an emulsion using 50 ng of lambda DNA and 50 ng of adapter. Lambda DNA was used as it is unlikely to form circles. Droplets were created by standard techniques (e.g., flow focusing at a T-junction using a PDMS-based microfluidic chip). Channel 1 contained DNA in ligase buffer (500 microliters) and channel 2 contained Quick Ligase in ligase buffer (500 microliters). PCR primers were designed to amplify internally within the lambda DNA (ligation-independent) or to amplify a portion of the adapter and the 5' or 3' end of the lambda DNA (ligation-dependent). Negative controls were performed in tubes to ensure ligation was ligase-dependent.
[0155] Figure 3 shows that ligation was achieved in both tubes and emulsion droplets. The forward primer for the adapter and the 5' primer for the lambda DNA only amplified in the presence of ligase, indicating that the adapter and the 5' end of the lambda DNA had ligated together in both tubes and emulsion droplets. The same result was achieved using the reverse primer for the adapter and the 3' primer for the lambda DNA, indicating that the adapter and the 3' end of the lambda DNA had ligated together in both tubes and emulsion droplets. These results demonstrate that ligation can be successfully performed in emulsion droplets.
Generation of end-labeled genomic DNA fragments
[0156] A method for bioinformatically associating the ends of genomic DNA is outlined in Figure 7. Genomic DNA is fragmented and size selected to a known size using techniques known in the art as described in Example 1. The genomic DNA is then A-tailed and ligated to a biotinylated, T-tailed asymmetric oligonucleotide adapter using methods well known in the art as described in Example 1.
[0157] Multiple droplet libraries (preferably 2-4 libraries) are created such that each library contains approximately 1000 unique double-stranded oligonucleotide indexes, thus approximately 2000-4000 unique indexes are used. For a given library, each droplet will contain many copies of one unique double-stranded index. Droplets may contain some or all of the key components of a ligation reaction (e.g., MgCl2, ATP, Ligase).
[0158] Fragmented genomic DNA ligated to an adapter is diluted to a desired concentration to control the number of molecules per droplet (e.g., a single DNA molecule per droplet or more than a single DNA molecule per droplet) and merged with the first index droplet library (Droplet Library "A" in Figure 4).
[0159] A ligation reaction is performed in each droplet, joining each unique double-stranded oligonucleotide index to the adapter on each end of the genomic DNA. The emulsion is then broken and the DNA is phosphorylated so that a second index can be ligated to the end of the first index.
[0160] The process of fragmented genomic DNA dilution, merging with a droplet library (e.g. Droplet Library "B" or "C" in Figure 4), ligation, breaking the droplets, and phosphorylation is repeated for the desired number of cycles, each time adding one new unique oligonucleotide index sequence to both ends of the fragmented genomic DNA.
[0161] After the final index addition, the result is fragmented genomic DNA uniquely end- labeled on both the 5' and 3' end with a unique label made up of many oligonucleotide indexes. The uniquely end-labeled fragmented genomic is then further fragmented and the ends are collected via streptavidin beads, which recognized the biotin label on the adapter. Fragments can be ligated to technology specific sequencing adapters (e.g., Illumina adapters) and sequenced. Ends are informatically paired by matching the unique label on one fragment of DNA with the same unique label on the other fragment of DNA as described in Example 1.
[0162] As with Example 1, this method can be used for other types of nucleic acids, such as RNA, cDNA, or PC -amplified DNA, or any other type of construct where such a labeling scheme is required
Validation of ligation-mediated end-labeling
[0163] Three libraries were created "in bulk" in microcentrifuge tubes from fragmented, end- repaired, A-tailed E. coli genomic DNA. For all three libraries, an initial ligation reaction was performed to add on a generic adapter to the ends of the E. coli genomic DNA. Genomic DNA libraries were then subjected to 1 (Library 1), 2 (Library 2), or 3 (Library 3) rounds of index ligation. Index ligation was performed by joining unique double-stranded oligonucleotide indexes to the adapter on each end of the genomic DNA. If required, the DNA was phosphorylated so that a second index could ligated to the end of the first index (two rounds of index ligation) or a third index could be ligated to the end of a second index (three rounds of index ligation). For round 1 and round 3 of index ligation, the same library/pool of indexes was used (pool A). For round 2, a library/pool of different indexes was used (pool B). As a final step, Illumina indexed adapters were ligated to all three genomic DNA libraries. Libraries were then pooled and sequence on an Illumina MiSeq (Illumina, San Diego, CA) using standard Illumina sequencing primers. Paired reads were identified and analyzed en masse (i.e. data from read 1 (3' end read) and read 2 (5' end read) was analyzed together as a single population). Sequencing data was analyzed by breaking up the reads into four separate, linear 8-mer populations (i.e. positions 1 through 4 in the read), since the indexes were each 8bp in length. For each position, the number of reads containing index or adapter were measured.
[0164] Figures 13 and 14 depict the results of the total read population analysis {en masse analysis) of the index ligation method. Library 1, which underwent 1 round of index ligation, had an expected outcome of an index read in position 1 and an adapter read in position 2. Library 2, which underwent 2 rounds of index ligation, had an expected outcome of an index read in position 1 and 2 and an adapter read in position 3. Library 3, which underwent 3 rounds of index ligation, had an expected outcome of an index read in position 1 to 3 and an adapter read in position 4. [0165] Figure 15 depicts the results of read pair analysis of individual molecules that underwent the index ligation method. Instead of analyzing the data from read 1 (3' end read) and read 2 (5' end read) together, reads were paired so that a molecule-by-molecule analysis was performed. First, reads were paired based on their unique read identifier. Each read was then broken down into 4 positions (8-mers) per read as described above. For each library, the total number of read pairs and the total number of unique molecular outcomes were determined and are shown in Figure 5 (Figure N). The composition of the top 10 most prevalent molecular outcomes and the number of pairs for each outcome are also shown in Figure 5 (Figure N). It was determined that the most desired outcome (the correct expected outcome) occurred 6% of the time in Library 1, 4% of the time in Library 2, and 4% of the time in Library 3.
[0166] Thus, Figures 13-15 show that the expected outcome was achieved and thus index ligation was a valid method of generating a unique label.
Example 3: Fragment Amplification
[0167] To increase the number of read pairs properly mated via their unique index combination, three methods are proposed to maximize fragment end recovery. These methods utilize the fragment preparation and indexing techniques described above, but vary in their approach to recovering and amplifying fragment ends within the library construction process.
Transposome-based Selection and Amplification of Ends
[0168] As shown in Figure 22, DNA samples are sheared to a desired size then the "Cap" and random combinations of index sequences are symmetrically attached to the fragment ends via ligation. Following the final round of index ligation, a new adapter containing an Illumina sequencing primer (SP1) adjacent to the Illumina P7 sequence is attached to the ends of the molecules via ligation as described above. The population of molecules is then incubated in the presence of a transposome carrying a different Illumina sequencing primer (SP2) adjacent to the Illumina P5 sequence. This reaction creates many fragments where both ends are flanked by the Illumina P5 sequence, but only two fragments per molecule that carry both the Illumina P7 and P5 sequences. PCR amplification using primers to P5/P7 is performed in order to enrich/select the fragment ends. Enrichment of Ends via/rc Vitro Transcription
[0169] As seen in Figures 23a and b, DNA samples are sheared to a desired size then the Cap and random combinations of index sequences are symmetrically attached to the fragment ends via ligation. Following the final round of index ligation, a new adapter sequence containing an Illumina sequencing primer (SP1) adjacent to an optimized T7 RNA polymerase promoter is attached to the ends of the molecules via ligation as described above. In vitro transcription (IVT) via T7 RNA polymerase is then performed in order to amplify both ends of a given molecule. Following IVT, a primer containing a random nucleotide sequence of a set length (i.e., pentamer, hexamer, etc.) flanked by a different Illumina sequencing primer (SP2) is utilized as the primer in a reverse transcription reaction. Alternatively, RNA molecules may be trimmed to a desired size range and ligated to the Illumina sequencing primer (SP2) via standard techniques. Illumina P5 and P7 sites are then added to the cDNA via PCR using primers carrying Illumina P5-SP1 and P7-SP2 sequences.
Amplification of Ends via Anchored PCR
[0170] As shown in Figure 24, DNA samples are sheared to a desired size then the Cap and random combinations of index sequences are symmetrically attached to the fragment ends via ligation. Following the final round of index ligation, a new adapter containing an Illumina sequencing primer (SP1) adjacent to the Illumina P7 sequence is attached to the ends of the molecules via ligation as described above. The population of molecules is then incubated in the presence of Fragmentase or a cocktail of restriction endonucleases to liberate the ends of the molecules. Fragments are then tailed at the 3' end using terminal transferase to attach a set number of specific nucleotides to the fragment ends, effectively creating a common priming sequence on the ends of all molecules. Alternatively, priming sequences may be ligated to the 3' of the molecules using standard techniques. The fragments are then amplified via PCR using SP2-P7 and SP1-P5 primers where the SP1-P5 primer contains a tail complementary to the priming site attached in the previous step.
[0171] The invention will be further described by the following numbered paragraphs:
1. A method for labeling a nucleic acid at both its 5 ' and 3 ' ends with a unique label, comprising the steps of:
a) providing a pool of nucleic acids; and b) sequentially end-labeling said nucleic acids with a random combination of n detectable oligonucleotide tags, each of said oligonucleotide tags optionally comprising a cohesive overhang of x base pairs in length, wherein each detectable oligonucleotide tag is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of said nucleic acid,
wherein said method is performed in emulsion droplets, and
wherein each end-labeled nucleic acid is identically labeled at its 5 ' and 3 ' ends.
2. The method according to paragraph 1, wherein x is greater than about two base pairs.
3. The method according to paragraph 1, wherein x is from about two to about ten base pairs.
4. The method according to paragraph 1, wherein x is about four base pairs.
5. The method according to paragraph 1, wherein said detectable oligonucleotide tag is from about 10 to about 20 base pairs in length.
6. The method according to paragraph 3, wherein said oligonucleotide tag is selected from a tag in Table 1.
7. The method according to paragraph 1, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 , 103, 104, 105, or 106 or more detectable oligonucleotide tags.
8. A method, comprising:
sequentially attaching at least two detectable oligonucleotide tags to a 5' and/or 3' end a first nucleic acid, wherein each detectable oligonucleotide tag is randomly selected from a plurality of detectable oligonucleotide tags, thereby generating a second nucleic acid comprising the first nucleic acid attached at its 5' and/or 3' end with a unique combination of detectable oligonucleotide tags, wherein the plurality of second nucleic acids is generated using emulsion droplets.
9. The method of paragraph 8, wherein the first nucleic acid is a genomic DNA fragment.
10. The method of paragraph 9, wherein the second nucleic acid is a genomic DNA fragment attached to the unique combination of detectable oligonucleotide tags at its 5' or 3' end. 11. The method of paragraph 9, wherein the second nucleic acid is a genomic DNA fragment attached to the same unique combination of detectable oligonucleotide tags at its 5' and 3' end.
12. The method of paragraph 9, further comprising fragmenting the second nucleic acid.
13. The method of paragraph 8, wherein sequentially attaching the at least two detectable oligonucleotide tags to the first nucleic acid comprises ligation, polymerization, or a combination thereof.
14. A method, comprising:
(a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acid;
(b) fusing each individual library droplet with a single index droplet from a plurality of mi index droplets, each index droplet comprising a plurality of one unique detectable oligonucleotide tag;
(c) end-labeling the nucleic acid with the unique detectable oligonucleotide tag in a fused droplet;
(d) harvesting end-labeled nucleic acids from the fused droplets and generating another population of library droplets comprising end-labeled nucleic acids;
(e) repeating steps (b) to (d) n times to produce nucleic acids end-labeled with n unique detectable oligonucleotide tag, wherein the n unique detectable oligonucleotide tags generate an (mi)(m2)(m3)...(mn) number of combinations that is greater than the number of starting nucleic acids; and
(f) amplifying the end-labeled nucleic acid formed in step (e).
15. The method of any one of paragraphia wherein end-labeling comprises ligation of the unique oligonucleotide tag with the nucleic acid.
16. The method of paragraph 15, wherein the unique oligonucleotide tag is double- stranded.
17. The method of according to paragraph 14, further comprising phosphorylating the nucleic acids between steps (b) and (c).
18. The method of according to paragraph 14, wherein end-labeling comprises a polymerase-mediated fill-in reaction. 19. The method of paragraph 18, wherein the polymerase-mediated fill-in reaction comprises:
(a) producing a single-stranded cohesive overhang on the nucleic acid, wherein the cohesive overhang is complementary to one end of the unique detectable oligonucleotide tag;
(b) annealing the complementary end of the unique oligonucleotide tag to the single-stranded cohesive overhang such that at least one nucleotide of the unique detectable oligonucleotide tag is not annealed to the nucleic acid, producing a unique detectable oligonucleotide tag cohesive overhang; and
(c) extending the single-stranded cohesive overhang of (a) using a polymerase and nucleotides complementary to the unique detectable oligonucleotide tag cohesive overhang to produce a double-stranded unique detectable oligonucleotide tag.
20. The method of paragraph 19, wherein the single-stranded cohesive overhang on the nucleic acid is produced by a USER enzyme.
21. The method of accroding to paragraph 19, wherein the unique detectable oligonucleotide tag is single-stranded.
22. The method of according to paragraph 19, wherein an oligonucleotide adapter is added to the nucleic acids before labeling with the unique detectable oligonucleotide tags.
23. The method of paragraph 20, wherein the adapter comprises biotin.
24. The method of paragraph 20, wherein the adapter comprises a thymidine tail cohesive overhang.
25. The method of paragraph 19, wherein labeling occurs at the 5' and 3' ends of the nucleic acid.
26. The method of paragraph 19, wherein labeling occurs at the 5' or the 3' end of the nucleic acid.
27. A labeled nucleic acid obtainable by the method of paragraph 1.
28. The method of paragraph 15, wherein amplification step (f) comprises the steps of:
(i) attaching an adapter comprising a first sequencing primer to said nucleic acid; (ii) incubating said nucleic acid in the presence of a transposome comprising a second sequencing primer; and
(iii) performing PCR amplification so as to amplify the ends of said nucleic acid.
29. The method of paragraph 14, wherein amplification step (f) comprises the steps of:
(i) attaching an adapter comprising a first sequencing primer to said nucleic acid;
(ii) performing in vitro transcription using a RNA polymerase;
(iii) performing a reverse transcription using a primer comprising a random nucleotide sequence of a given length flanked by a second sequencing primer or performing a reverse transcription using a primer comprising a nucleotide sequence attached to the 3' end of the nucleic acid; and
(iv) performing PCR amplification so as to amplify the ends of said nucleic acid.
30. The method of paragraph 14, wherein amplification step (f) comprises the steps of:
(i) attaching an adapter comprising a first sequencing primer to said nucleic acid;
(ii) incubating said nucleic acid in fragmentase or a combination of one or more restriction endonucleases so as to liberate the ends of said nucleic acid thereby forming fragments;
(iii) attaching a given number of specific nucleotides to the ends of said fragments; and
(iv) performing PCR amplification on the fragments formed in step (iii) using a second sequencing primer.
* A A
[0172] It is to be understood that the invention is not limited to the particular embodiments of the invention described above, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.

Claims

1. A method for labeling a nucleic acid at both its 5 ' and 3 ' ends with a unique label, comprising the steps of:
a) providing a pool of nucleic acids; and
b) sequentially end-labeling said nucleic acids with a random combination of n detectable oligonucleotide tags, each of said oligonucleotide tags optionally comprising a cohesive overhang of x base pairs in length, wherein each detectable oligonucleotide tag is randomly and independently selected from a number of detectable oligonucleotide tags that is less than the number of nucleic acids, and n is the number of oligonucleotides attached to an end of said nucleic acid,
wherein said method is performed in emulsion droplets, and
wherein each end-labeled nucleic acid is identically labeled at its 5 ' and 3 ' ends.
nucleic acid
2. The method according to claim 1 , wherein x is greater than about two base pairs.
3. The method according to claim 1, wherein x is from about two to about ten base pairs.
4. The method according to claim 1, wherein x is about four base pairs.
5. The method according to claim 1, wherein said detectable oligonucleotide tag is from about 10 to about 20 base pairs in length.
6. The method according to claim 3, wherein said oligonucleotide tag is selected from a tag in Table 1.
7. The method according to claim 1, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 10 2, 103J, 104, 105, or 106 or more detectable oligonucleotide tags.
8. A method, comprising:
sequentially attaching at least two detectable oligonucleotide tags to a 5' and/or 3' end a first nucleic acid, wherein each detectable oligonucleotide tag is randomly selected from a plurality of detectable oligonucleotide tags, thereby generating a second nucleic acid comprising the first nucleic acid attached at its 5' and/or 3' end with a unique combination of detectable oligonucleotide tags, wherein the plurality of second nucleic acids is generated using emulsion droplets.
9. The method of claim 8, wherein the first nucleic acid is a genomic DNA fragment.
10. The method of claim 9, wherein the second nucleic acid is a genomic DNA fragment attached to the unique combination of detectable oligonucleotide tags at its 5' or 3' end.
11. The method of claim 9, wherein the second nucleic acid is a genomic DNA fragment attached to the same unique combination of detectable oligonucleotide tags at its 5' and 3' end.
12. The method of claim 9, further comprising fragmenting the second nucleic acid.
13. The method of claim 8, wherein sequentially attaching the at least two detectable oligonucleotide tags to the first nucleic acid comprises ligation, polymerization, or a combination thereof.
14. A method, comprising :
(a) providing a population of library droplets comprising nucleic acids, wherein each droplet comprises a nucleic acid;
(b) fusing each individual library droplet with a single index droplet from a plurality of ni! index droplets, each index droplet comprising a plurality of one unique detectable oligonucleotide tag;
(c) end-labeling the nucleic acid with the unique detectable oligonucleotide tag in a fused droplet;
(d) harvesting end-labeled nucleic acids from the fused droplets and generating another population of library droplets comprising end-labeled nucleic acids;
(e) repeating steps (b) to (d) n times to produce nucleic acids end-labeled with n unique detectable oligonucleotide tag, wherein the n unique detectable oligonucleotide tags generate an (m1)(m2)(m3)...(mn) number of combinations that is greater than the number of starting nucleic acids; and
(f) amplifying the end-labeled nucleic acid formed in step (e).
15. The method of any one of claiml4, wherein end-labeling comprises ligation of the unique oligonucleotide tag with the nucleic acid.
16. The method of claim 15, wherein the unique oligonucleotide tag is double- stranded.
17. The method of according to claim 14, further comprising phosphorylating the nucleic acids between steps (b) and (c).
18. The method of according to claim 14, wherein end-labeling comprises a polymerase-mediated fill-in reaction.
19. The method of claim 18, wherein the polymerase-mediated fill-in reaction comprises:
(a) producing a single-stranded cohesive overhang on the nucleic acid, wherein the cohesive overhang is complementary to one end of the unique detectable oligonucleotide tag;
(b) annealing the complementary end of the unique oligonucleotide tag to the single-stranded cohesive overhang such that at least one nucleotide of the unique detectable oligonucleotide tag is not annealed to the nucleic acid, producing a unique detectable oligonucleotide tag cohesive overhang; and
(c) extending the single-stranded cohesive overhang of (a) using a polymerase and nucleotides complementary to the unique detectable oligonucleotide tag cohesive overhang to produce a double-stranded unique detectable oligonucleotide tag.
20. The method of claim 19, wherein the single-stranded cohesive overhang on the nucleic acid is produced by a USER enzyme.
21. The method of accroding to claim 19, wherein the unique detectable oligonucleotide tag is single-stranded.
22. The method of according to claim 19, wherein an oligonucleotide adapter is added to the nucleic acids before labeling with the unique detectable oligonucleotide tags.
23. The method of claim 20, wherein the adapter comprises biotin.
24. The method of claim 20, wherein the adapter comprises a thymidine tail cohesive overhang.
25. The method of claim 19, wherein labeling occurs at the 5' and 3' ends of the nucleic acid.
26. The method of claim 19, wherein labeling occurs at the 5' or the 3' end of the nucleic acid.
27. A labeled nucleic acid obtainable by the method of claim 1.
28. The method of claim 15, wherein amplification step (f) comprises the steps of:
(i) attaching an adapter comprising a first sequencing primer to said nucleic acid;
(ii) incubating said nucleic acid in the presence of a transposome comprising a second sequencing primer; and
(iii) performing PCR amplification so as to amplify the ends of said nucleic acid.
29. The method of claim 14, wherein amplification step (f) comprises the steps of:
(i) attaching an adapter comprising a first sequencing primer to said nucleic acid;
(ii) performing in vitro transcription using a RNA polymerase;
(iii) performing a reverse transcription using a primer comprising a random nucleotide sequence of a given length flanked by a second sequencing primer or performing a reverse transcription using a primer comprising a nucleotide sequence attached to the 3' end of the nucleic acid; and
(iv) performing PCR amplification so as to amplify the ends of said nucleic acid.
30. The method of claim 14, wherein amplification step (f) comprises the steps of:
(i) attaching an adapter comprising a first sequencing primer to said nucleic acid;
(ii) incubating said nucleic acid in fragmentase or a combination of one or more restriction endonucleases so as to liberate the ends of said nucleic acid thereby forming fragments;
(iii) attaching a given number of specific nucleotides to the ends of said fragments; and
(iv) performing PCR amplification on the fragments formed in step (iii) using a second sequencing primer.
PCT/US2013/060990 2013-03-13 2013-09-20 Compositions and methods for long insert, paired end libraries of nucleic acids in emulsion droplets WO2014143157A1 (en)

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