WO2016077602A1 - Méthodes de séquençage de nouvelle génération - Google Patents

Méthodes de séquençage de nouvelle génération Download PDF

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WO2016077602A1
WO2016077602A1 PCT/US2015/060414 US2015060414W WO2016077602A1 WO 2016077602 A1 WO2016077602 A1 WO 2016077602A1 US 2015060414 W US2015060414 W US 2015060414W WO 2016077602 A1 WO2016077602 A1 WO 2016077602A1
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sequencing
nucleic acid
reaction
library
molecules
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PCT/US2015/060414
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Mark W. Eshoo
John M. Picuri
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Ibis Biosciences, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present disclosure provides methods, systems, kits, and compositions for performing sequencing reactions with limiting reagent concentrations.
  • the present disclosure provides methods of performing next generation sequencing reactions without quantifying sequencing templates (e.g., nucleic acid libraries).
  • the present disclosure provides methods, systems, kits, and compositions for performing sequencing reactions with limiting reagent concentrations.
  • the present disclosure provides methods of performing next generation sequencing reactions without quantifying sequencing templates (e.g., nucleic acid libraries).
  • the present disclosure provides a method of sequencing a nucleic acid library where the concentration of the library is unknown, comprising: a) contacting a nucleic acid library that has not been quantitated with a plurality of sequencing reaction components, wherein at least one of the reaction components (e.g., polymerase, dNTPs, or sequencing primers) is present at a limiting concentration; and b) performing the sequencing reaction.
  • the sequencing is next generation sequencing (e.g., single molecule sequencing).
  • the sequencing reaction is performed on a solid support comprising a plurality of reaction sites.
  • at least one of the reaction components is present at a concentration of one molecule of reaction component per reaction site.
  • the nucleic acid library is prepared by whole genome amplification. In some embodiments, the whole genome amplification is performed on genomic DNA with at least one amplification reagent present in limiting quantities, and wherein the concentration of the genomic DNA is unknown. In some embodiments, the nucleic acid library has or is suspected of having at least 10,000 (e.g., at least 100,000, at least 1,000,000 or more) target sequences.
  • Additional embodiments provide a system comprising: a) a container consisting of, consisting essentially of, or comprising a nucleic acid sequencing library that has not been quantitated and a plurality of reaction components, wherein at least one of the reaction components is present at limiting concentration; and b) a nucleic acid sequencing device or system.
  • a system comprising: a) a container consisting of, consisting essentially of, or comprising a first number of molecules of sequencing library nucleic acid targets and a second number of polymerase molecules, wherein the second number of molecules is less than the first number of molecules (e.g., 2 times, 4 time, 10 times, 100 times less or lower); and b) a nucleic acid sequencing device or system.
  • Figure 1 shows a schematic of a prior art preparation where a DNA library is quantitated prior to sequencing, and none of the reagents in the sequencing reaction are provided in a limiting amount.
  • Figure 2 shows an exemplary embodiment of the present disclosure where no DNA library quantitation is conducted (and no dilution series are prepared), and a specific limiting amount of a sequencing component is provided (e.g., polymerase in this figure) such that only productive sequencing complexes are formed prior to sequencing.
  • a sequencing component e.g., polymerase in this figure
  • Figure 3 shows the number of sequencing reads versus library concentration.
  • Figure 4 shows normalization of input into library preparation process by limitation of a key reaction component.
  • a 1 ,000,000 fold difference in input amount 100 ng to 100 fg was normalized to within 5 fold output (10 ⁇ g to 2 ⁇ g) by limiting the amount of dNTPs used in the reaction. This allows a large range of input concentrations to be successfully used with the library preparation process without the need for quantification. Changing the dNTP concentration and time allows for adjustment of the output amount created.
  • amplifying or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable.
  • Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR) are forms of amplification.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Amplification is not limited to the strict duplication of the starting molecule.
  • the generation of multiple cDNA molecules from a limited amount of RNA in a sample using reverse transcription (RT)-PCR is a form of amplification.
  • the generation of multiple RNA molecules from a single DNA molecule during the process of transcription is also a form of amplification.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (e.g. , in the presence of nucleotides and an inducing agent such as a biocatalyst (e.g. , a DNA polymerase or the like) and at a suitable temperature and H).
  • the primer is typically single stranded for maximum efficiency in amplification, but may alternatively be double stranded.
  • the primer is generally first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • sample refers to anything capable of being analyzed by the methods provided herein that is suspected of containing a target nucleic acid sequence.
  • Samples may be complex samples or mixed samples, which contain nucleic acids comprising multiple different nucleic acid sequences. Samples may comprise nucleic acids from more than one source (e.g. difference species, different subspecies, etc.), subject, and/or individual.
  • the methods provided herein comprise purifying the sample or purifying the nucleic acid(s) from the sample.
  • the sample contains purified nucleic acid.
  • a sample is derived from a biological, clinical, environmental, research, forensic, or other source.
  • the present disclosure provides methods, systems, kits, and compositions for performing sequencing reactions with limiting reagent concentrations.
  • the present disclosure provides methods of performing next generation sequencing reactions without quantifying sequencing templates (e.g., nucleic acid libraries).
  • the systems and methods of the present disclosure eliminate the need to quantitate sequencing libraries prior to performing next generation sequencing by limiting an element of the sequencing reaction (e.g., polymerase or primers). This is important because currently all next generation sequencing technologies require very accurate quantitation of the sequencing libraries prior to sequencing, which adds significant time and labor to the next generation sequencing process and makes integration of the workflow (e.g., in a microfluidic device) significantly more complex and expensive. Additionally, even after this laborious quantitation process, current technologies still often require further empirical validation (for example a dilution series of library) of this quantitation prior to full scale sequencing.
  • an element of the sequencing reaction e.g., polymerase or primers
  • Embodiments of the present disclosure avoid the need to quantitate sequencing templates (e.g., libraries) prior to sequencing by limiting one or more reagents of the sequencing reaction (e.g., nucleic acid polymerase, sequencing primers, nucleotides, etc.). This controls the number of molecules that are being sequenced and assures that there are an optima and known number or transcripts being sequenced.
  • sequencing templates e.g., libraries
  • reagents of the sequencing reaction e.g., nucleic acid polymerase, sequencing primers, nucleotides, etc.
  • the methods of the present disclosure achieve the same sequencing performance using less time, less labor and at a lower cost. Additionally, the complexity of the device/instrumentation needed to perform the integration of the sequencing workflow is significantly reduced.
  • the present disclosure finds use in a variety of sequencing techniques (e.g., next generation sequencing).
  • the sequencing nucleic acid library has or is suspected of having at least 10,000 (e.g., at least 100,000, at least 1,000,000 or more) target sequences.
  • sequencing methods are single molecule sequencing methods.
  • it is optimal to have one molecule of DNA per limiting reagent (e.g., polymerase).
  • the amount of polymerase or other reagent is limited so that there is less reagent than DNA.
  • the amount of polymerase is optimized such that one molecule of polymerase or primer per reaction site (e.g., on a solid support) is provided.
  • the sequencing is the real-time single molecule sequencing system developed by Pacific Biosciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 7,170,050; U.S. Pat. No. 7,302,146; U.S. Pat. No. 7,313,308; U.S. Pat. No. 7,476,503; all of which are herein incorporated by reference) utilizes reaction wells 50-100 nm in diameter and encompassing a reaction volume of approximately 20
  • zeptoliters (10 x 10 " L). Sequencing reactions are performed using immobilized template, modified phi29 DNA polymerase, and high local concentrations of fluorescently labeled dNTPs. High local concentrations and continuous reaction conditions allow incorporation events to be captured in real time by fluor signal detection using laser excitation, an optical waveguide, and a CCD camera.
  • the single molecule real time (SMRT) DNA sequencing methods using zero-mode waveguides (ZMWs) developed by Pacific Biosciences, or similar methods are employed.
  • ZMWs zero-mode waveguides
  • DNA sequencing is performed on SMRT chips, each containing thousands of zero-mode waveguides (ZMWs).
  • a ZMW is a hole, tens of nanometers in diameter, fabricated in a lOOnm metal film deposited on a silicon dioxide substrate.
  • Each ZMW becomes a nanophotonic visualization chamber providing a detection volume of just 20 zeptoliters (10-21 liters). At this volume, the activity of a single molecule can be detected amongst a background of thousands of labeled nucleotides.
  • the ZMW provides a window for watching DNA polymerase as it performs sequencing by synthesis.
  • a single DNA polymerase molecule is attached to the bottom surface such that it permanently resides within the detection volume.
  • Phospho linked nucleotides each type labeled with a different colored fluorophore, are then introduced into the reaction solution at high concentrations which promote enzyme speed, accuracy, and processivity. Due to the small size of the ZMW, even at these high, biologically relevant concentrations, the detection volume is occupied by nucleotides only a small fraction of the time. In addition, visits to the detection volume are fast, lasting only a few microseconds, due to the very small distance that diffusion has to carry the nucleotides. The result is a very low background.
  • NUCLEOTIDES entitled “Substrates, systems and methods for analyzing materials”
  • 20080152280 entitled “Substrates, systems and methods for analyzing materials”
  • 20080145278 entitled “Uniform surfaces for hybrid material substrates and methods for making and using same”
  • 20070238679 entitled “Articles having localized molecules disposed thereon and methods of producing same”
  • 20070231804 entitled “Methods, systems and compositions for monitoring enzyme activity and applications thereof
  • 20070206187 entitled “Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources”
  • 20070196846 entitled “Polymerases for nucleotide analogue incorporation”
  • 20070188750 entitled “Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources”
  • 20070161017 entitled “MITIGATION OF PHOTODAMAGE IN ANALYTICAL REACTIONS", 20070141598, entitled “Nucleotide Compositions and Uses Thereof, 20070134128, entitled “Uniform surfaces for hybrid material substrate and methods for making and using same", 20070128133, entitled “Mitigation of photodamage in analytical reactions", 20070077564, entitled “Reactive surfaces, substrates and methods of producing same", 20070072196, entitled “Fluorescent nucleotide analogs and uses therefore", and 20070036511, entitled “Methods and systems for monitoring multiple optical signals from a single source”, and Korlach et al.
  • DNA sequencing techniques can be used, including fluorescence-based sequencing methodologies (See, e.g., Birren et al, Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety).
  • automated sequencing techniques understood in that art are utilized.
  • DNA sequencing is achieved by parallel oligonucleotide extension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., and U.S. Pat. No. 6,306,597 to Macevicz et al., both of which are herein incorporated by reference in their entireties).
  • sequencing techniques include the Church polony technology (Mitra et al, 2003, Analytical Biochemistry 320, 55-65; Shendure et al, 2005 Science 309, 1728-1732; U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; herein incorporated by reference in their entireties) the 454 picotiter pyrosequencing technology (Margulies et al, 2005 Nature 437, 376-380; US 20050130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat.
  • chain terminator sequencing is utilized.
  • Chain terminator sequencing uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive, or other labeled, oligonucleotide primer complementary to the template at that region.
  • the oligonucleotide primer is extended using a DNA polymerase, standard four deoxynucleotide bases, and a low concentration of one chain terminating nucleotide, most commonly a di- deoxynucleotide. This reaction is repeated in four separate tubes with each of the bases taking turns as the di-deoxynucleotide.
  • the DNA polymerase Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular di- deoxynucleotide is used.
  • the fragments are size-separated by electrophoresis in a slab polyacrylamide gel or a capillary tube filled with a viscous polymer. The sequence is determined by reading which lane produces a visualized mark from the labeled primer as you scan from the top of the gel to the bottom.
  • Dye terminator sequencing alternatively labels the terminators. Complete sequencing can be performed in a single reaction by labeling each of the di- deoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength.
  • NGS Next- generation sequencing
  • Non-amplification approaches also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos Biosciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., and Pacific Biosciences, respectively.
  • template DNA is fragmented, end-repaired, ligated to adaptors, and clonally amplified in-situ by capturing single template molecules with beads bearing oligonucleotides complementary to the adaptors.
  • Each bead bearing a single template type is compartmentalized into a water-in-oil microvesicle, and the template is clonally amplified using a technique referred to as emulsion PCR.
  • the emulsion is disrupted after amplification and beads are deposited into individual wells of a picotitre plate functioning as a flow cell during the sequencing reactions. Ordered, iterative introduction of each of the four dNTP reagents occurs in the flow cell in the presence of sequencing enzymes and luminescent reporter such as luciferase.
  • sequencing data are produced in the form of shorter- length reads.
  • single-stranded fragmented DNA is end-repaired to generate 5'-phosphorylated blunt ends, followed by Klenow-mediated addition of a single A base to the 3' end of the fragments.
  • Klenow-mediated addition facilitates addition of T- overhang adaptor oligonucleotides, which are subsequently used to capture the template-adaptor molecules on the surface of a flow cell that is studded with oligonucleotide anchors.
  • the anchor is used as a PCR primer, but because of the length of the template and its proximity to other nearby anchor oligonucleotides, extension by PCR results in the "arching over" of the molecule to hybridize with an adjacent anchor oligonucleotide to form a bridge structure on the surface of the flow cell.
  • These loops of DNA are denatured and cleaved. Forward strands are then sequenced with reversible dye terminators.
  • the sequence of incorporated nucleotides is determined by detection of post-incorporation fluorescence, with each fluor and block removed prior to the next cycle of dNTP addition. Sequence read length ranges from 36 nucleotides to over 50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.
  • Sequencing nucleic acid molecules using SOLiD technology also involves fragmentation of the template, ligation to oligonucleotide adaptors, attachment to beads, and clonal amplification by emulsion PCR.
  • beads bearing template are immobilized on a derivatized surface of a glass flow-cell, and a primer complementary to the adaptor oligonucleotide is annealed.
  • a primer complementary to the adaptor oligonucleotide is annealed.
  • this primer is instead used to provide a 5' phosphate group for ligation to
  • interrogation probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels.
  • interrogation probes In the SOLiD system, interrogation probes have 16 possible combinations of the two bases at the 3' end of each probe, and one of four fluors at the 5' end. Fluor color and thus identity of each probe corresponds to specified color-space coding schemes. Multiple rounds (usually 7) of probe annealing, ligation, and fluor detection are followed by denaturation, and then a second round of sequencing using a primer that is offset by one base relative to the initial primer. In this manner, the template sequence can be computationally reconstructed, and template bases are interrogated twice, resulting in increased accuracy. Sequence read length averages 35 nucleotides, and overall output exceeds 4 billion bases per sequencing run.
  • nanopore sequencing in employed (see, e.g., Astier et al, J Am Chem Soc. 2006 Feb 8;128(5): 1705-10, herein incorporated by reference).
  • the theory behind nanopore sequencing has to do with what occurs when the nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it: under these conditions a slight electric current due to conduction of ions through the nanopore can be observed, and the amount of current is exceedingly sensitive to the size of the nanopore. If DNA molecules pass (or part of the DNA molecule passes) through the nanopore, this can create a change in the magnitude of the current through the nanopore, thereby allowing the sequences of the DNA molecule to be determined.
  • HeliScope by Helicos Biosciences is employed (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat. No.
  • Template DNA is fragmented and polyadenylated at the 3' end, with the final adenosine bearing a fluorescent label.
  • Denatured polyadenylated template fragments are ligated to poly(dT) oligonucleotides on the surface of a flow cell.
  • Sequencing is achieved by addition of polymerase and serial addition of fluorescently-labeled dNTP reagents. Incorporation events result in fluor signal corresponding to the dNTP, and signal is captured by a CCD camera before each round of dNTP addition. Sequence read length ranges from 25-50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.
  • the Ion Torrent technology (Life Technologies) is employed to sequence purified target nucleic acid sequences.
  • the Ion Torrent technology is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA (see, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appl. Pub. Nos. 20090026082, 20090127589, 20100301398, 20100197507, 20100188073, and 20100137143, incorporated by reference in their entireties for all purposes).
  • a microwell contains a template DNA strand to be sequenced. Beneath the layer of microwells is a hypersensitive ISFET ion sensor.
  • CMOS semiconductor chip all layers are contained within a CMOS semiconductor chip, similar to that used in the electronics industry.
  • a dNTP When a dNTP is incorporated into the growing complementary strand a hydrogen ion is released, which triggers a hypersensitive ion sensor. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.
  • This technology differs from other sequencing technologies in that no modified nucleotides or optics are used.
  • the per-base accuracy of the Ion Torrent sequencer is -99.6% for 50 base reads, with -100 Mb generated per run. The read-length is 100 base pairs. The accuracy for homopolymer repeats of 5 repeats in length is -98%.
  • the benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs.
  • the sequencing process typically includes providing a daughter strand produced by a template-directed synthesis.
  • the daughter strand generally includes a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid in which the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond.
  • the selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand.
  • the Xpandomer typically includes the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Additional details relating to Xpandomer-based approaches are described in, for example, U.S. Patent Publication No. 20090035777.
  • the target nucleic acid sequences are subjected to amplification (e.g., prior to sequencing or during library preparation).
  • amplification is whole genome amplification.
  • Exemplary amplification reactions include, but are not limited to the polymerase chain reaction (PCR) or ligase chain reaction (LCR), each of which is driven by thermal cycling.
  • Amplifications used in method or assays of the present disclosure may be performed in bulk and/or partitioned volumes (e.g. droplets).
  • Alternative amplification reactions which may be performed isothermally, also find use herein, such as branched-probe DNA assays, cascade -RCA, helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nucleic acid based amplification (NASBA), nicking enzyme amplification reaction (NEAR), PAN- AC, Q-beta replicase amplification, rolling circle replication (RCA), self-sustaining sequence replication, strand-displacement amplification, and the like.
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid based amplification
  • NEAR nicking enzyme amplification reaction
  • PAN- AC Q-beta replicase amplification
  • RCA rolling circle replication
  • self-sustaining sequence replication strand-displacement amplification, and the like.
  • Amplification may be performed with any suitable reagents (e.g. template nucleic acid (e.g. DNA or RNA), primers, probes, buffers, replication catalyzing enzyme (e.g. DNA polymerase, RNA polymerase), nucleotides, salts (e.g. MgCl 2 ), etc.
  • reagents e.g. template nucleic acid (e.g. DNA or RNA), primers, probes, buffers, replication catalyzing enzyme (e.g. DNA polymerase, RNA polymerase), nucleotides, salts (e.g. MgCl 2 ), etc.
  • an amplification mixture includes any combination of at least one primer or primer pair, at least one probe, at least one replication enzyme
  • dNTPs and/or NTPs deoxynucleotide triphosphates
  • the present disclosure utilizes nucleic acid
  • amplification that relies on alternating cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication (e.g., PCR).
  • thermal cycling i.e., thermal cycling
  • PCR is used to amplify target nucleic acids (e.g. partitioned targets). PCR may be performed by thermal cycling between two or more temperature set points, such as a higher melting (denaturation) temperature and a lower
  • PCR may be performed with a thermostable polymerase, such as Taq DNA polymerase (e.g., wild-type enzyme, a Stoffel fragment, FastStart polymerase, etc.), Pfu DNA polymerase, S-Tbr polymerase, Tth polymerase, Vent polymerase, or a combination thereof, among others.
  • Typical PCR methods produce an exponential increase in the amount of a product amplicon over successive cycles, although linear PCR methods also find use in the present disclosure.
  • Samples may be derived from any suitable source, and for purposes related to any field, including but not limited to diagnostics, research, forensics, epidemiology, pathology, archaeology, etc.
  • a sample may be biological,
  • Samples may include nucleic acid derived from any suitable source, including eukaryotes, prokaryotes (e.g. infectious bacteria), mammals, humans, non-human primates, canines, felines, bovines, equines, porcines, mice, viruses, etc. Samples may contain, e.g., whole organisms, organs, tissues, cells, organelles (e.g., chloroplasts, mitochondria), synthetic nucleic acid, cell lysate, etc. Nucleic acid present in a sample (e.g.
  • target nucleic acid may be of any type, e.g., genomic DNA, RNA, plasmids, bacteriophages, synthetic origin, natural origin, and/or artificial sequences (non-naturally occurring), synthetically-produced but naturally occurring sequences, etc.
  • Biological specimens may, for example, include whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal (CSF) fluids, amniotic fluid, seminal fluid, vaginal excretions, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluids, intestinal fluids, fecal samples, and swabs or washes (e.g., oral, nasopharangeal, optic, rectal, intestinal, vaginal, epidermal, etc.) and/or other biological specimens.
  • samples that find use with the present disclosure are mixed samples (e.g. containing mixed nucleic acid populations).
  • samples analyzed by methods herein contain, or may contain, a plurality of different nucleic acid sequences.
  • a sample e.g. mixed sample
  • contains one or more nucleic acid molecules e.g. 1... 10... 10 ... 10 3 ... 10 4 ... 10 5 ... 10 6 ... 10 7 , etc.
  • a sample e.g. mixed sample
  • a sample e.g. mixed sample
  • a sample e.g. mixed sample
  • a sample e.g. mixed sample
  • a sample e.g. mixed sample
  • a sample contains more nucleic acid molecules that do not contain a target sequence than nucleic acid molecules that do contain a target sequence (e.g. 1.01 : 1... 2: 1... 5: 1... 10: 1... 20: 1... 50: 1... 10 2 : 1... 10 3 : 1...10 4 :1 ... 10 5 : 1... 10 6 : 1... 10 7 : 1).
  • a sample contains more nucleic acid molecules that do contain a target sequence than nucleic acid molecules that do not contain a target sequence (e.g. 1.01 : 1... 2: 1... 5: 1... 10: 1... 20: 1... 50: 1... 10 2 : 1... 10 3 : 1...10 4 :1 ...
  • a sample contains a single target sequence which may be present in one or more nucleic acid molecules in the sample. In some embodiments, a sample contains two or more target sequences (e.g. 2, 3, 4, 5...10...20...50...100, etc.) which may each be present in one or more nucleic acid molecules in the sample.
  • target sequences e.g. 2, 3, 4, 5...10...20...50...100, etc.
  • Genomic DNA was carried through a library preparation protocol to create structurally linear, topologically circular DNA template for sequencing.
  • this procedure (1) amplified the DNA via a whole genome amplification (WGA) method (2) fragmented the DNA to an appropriate length using ultrasonic energy (3) prepared the fragmented DNA for ligation using a combination of polymerases and a kinase (4) attached sequencing adapters using a ligase (5) removed incomplete products using a combination of exonucleases (6) purified the sequencing library using a magnetic bead based purification method.
  • WGA whole genome amplification
  • This template was then annealed to a sequencing primer which is
  • the three concentrations of library (lx, 5x, lOx) all showed very similar average readlengths and read qualities.
  • the # of sequencing reads was slightly elevated (-50%) at the 5x and lOx library concentrations in comparison to the lx library concentration. (See Figure 3)
  • sequencing reads are the key metrics used to determine the success of a sequencing run.
  • read quality and average readlength remained essentially unchanged between the lx, 5x and lOx library concentrations, it is clear that changes in the library concentration have no effect when a key sequencing component (in this case polymerase) is limited.
  • Equal or improved performance at higher library to polymerase ratio indicates ability to load libraries within at least 10 fold concentration range by limiting an element of the sequencing reaction (e.g. polymerase).
  • reaction buffer in this case random septamers
  • primers in this case random septamers
  • polymerases in this case Phi 29 and Klenow exo-
  • an accessory enzyme in this case pyrophosphatase
  • dNTPs dNTPs
  • Figure 4 shows normalization of input into library preparation process by limitation of a reaction component. A 1,000,000 fold difference in input amount (100 ng to 100 fg) was normalized to within 5 fold output (10 ⁇ g to 2 ⁇ g) by limiting the amount of dNTPs used in the reaction. This allows a large range of input

Abstract

La présente invention concerne des procédés, des systèmes, des kits et des compositions permettant d'effectuer des réactions de séquençage en réduisant les concentrations en réactif. En particulier, la présente invention concerne des procédés permettant d'effectuer des réactions de séquençage de nouvelle génération sans quantification des modèles de séquençage (par exemple, des banques d'acides nucléiques).
PCT/US2015/060414 2014-11-12 2015-11-12 Méthodes de séquençage de nouvelle génération WO2016077602A1 (fr)

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