WO2016065298A1 - Systèmes, compositions et procédés pour la purification d'acide nucléique par taille - Google Patents

Systèmes, compositions et procédés pour la purification d'acide nucléique par taille Download PDF

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Publication number
WO2016065298A1
WO2016065298A1 PCT/US2015/057182 US2015057182W WO2016065298A1 WO 2016065298 A1 WO2016065298 A1 WO 2016065298A1 US 2015057182 W US2015057182 W US 2015057182W WO 2016065298 A1 WO2016065298 A1 WO 2016065298A1
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peg
nucleic acid
percentage
buffer
binding buffer
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PCT/US2015/057182
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English (en)
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Mark W. Eshoo
Alice T. HANG
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Ibis Biosciences, Inc.
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Publication of WO2016065298A1 publication Critical patent/WO2016065298A1/fr

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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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules

Definitions

  • kits used for generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other nucleic acid analysis technologies.
  • methods, compositions, and related kits for size selective nucleic acid purification of next generation sequencing libraries using paramagnetic beads with an optimal magnetic moment, a size selecting binding buffer, and a wash buffer compatible with automated micro fluidics are provided herein.
  • PEG Polyethylene Glycol
  • NaCl Sodium Chloride
  • kits used for generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other nucleic acid analysis technologies.
  • methods, compositions, and related kits for size selective nucleic acid purification of next generation sequencing libraries using paramagnetic beads with an optimal magnetic moment, a size selecting binding buffer, and a wash buffer compatible with microfluidics are provided herein.
  • the technology provides systems comprising a magnetic device capable of generating a three-dimensional magnetic field, a microfluidic cartridge device having one or more removable chambers for containing a biological sample, wherein the microfluidic cartridge is configured to be secured with magnetic device such that upon securing with the magnetic device the microfluidic cartridge device is exposed to a three- dimensional magnetic field generated with the pneumatic actuation device, and a binding buffer comprising PEG.
  • the percentage of PEG within the binding buffer is any percentage that facilitates optimal nucleic acid purification from a biological sample (e.g., where the purification is selective for DNA greater than 1000 base pairs). In some embodiments, the percentage of PEG within the binding buffer is between 6% and 4%. In some embodiments, the percentage of PEG within the binding buffer is between 5.14% and 4.18%. In some embodiments, the binding buffer further comprises nuclease free water and NaCl.
  • the systems further comprise solid supports comprising super paramagnetic beads coated with carboxylic acid functional groups.
  • the systems further comprise a washing buffer comprising
  • the percentage of PEG within the washing buffer is between 5% and 20%. In some embodiments, the percentage of PEG within the washing buffer is between 7.5%) and 16%>. In some embodiments, further comprises MgCl 2 and NaCl.
  • the microfluidic cartridge device is secured with the magnetic device.
  • the magnetic device is part of a pneumatic actuation device.
  • the systems further comprise a sequencing device.
  • the microfluidic cartridge device is engaged with the sequencing device.
  • the technology provides methods for purifying nucleic acid fragments, comprising mixing a sample comprising nucleic acid fragments with a binding buffer and solid supports, wherein the mixing results in a binding of the nucleic acid fragments with the solid supports, wherein the mixing occurs within a removable chamber secured with a microfluidic cartridge, wherein the microfluidic cartridge is secured with a magnetic device capable of generating a three-dimensional magnetic field, wherein the solid supports comprise super paramagnetic beads coated with carboxylic acid functional groups, wherein the binding buffer comprises PEG, and segregating the bound nucleic acid fragments through exposing the mixed sample to a three-dimensional magnetic field generated with the magnetic device.
  • the percentage of PEG within the binding buffer is any percentage that facilitates optimal nucleic acid purification from a biological sample (e.g., where the purification is selective for DNA greater than 1000 base pairs). In some embodiments, the percentage of PEG within the binding buffer is between 6% and 4%. In some embodiments, the percentage of PEG within the binding buffer is between 5.14% and 4.18%. In some embodiments, the binding buffer further comprises nuclease free water and NaCl.
  • the segregated nucleic acid fragments are primarily greater than 1000 base pairs (e.g., greater than 50% of the segregated nucleic acid fragments are over 1000 bps; greater than 70%> are over 1000 bps; greater than 80%> are over 1000 bps; greater than 90%> are over 1000 bps; greater than 95% are over 1000 bps; etc) (e.g., the average of the segregated nucleic acid fragments are over 1000 bps).
  • the methods further comprise washing the segregated nucleic acid fragments.
  • the washing comprises contacting the segregated nucleic acid fragments with a washing buffer comprising PEG, wherein the percentage of PEG within the washing buffer is between 5% and 20%.
  • the washing buffer further comprises MgCl 2 and NaCl. In some embodiments the percentage of PEG within the washing buffer is between 7.5% and 16%.
  • the methods further comprise eluting the segregated nucleic acid fragments.
  • the magnetic device is part of a pneumatic actuation device.
  • the methods further comprise delivering the segregated nucleic acid fragments to a sequencing device (e.g., a next generation sequencing device).
  • a sequencing device e.g., a next generation sequencing device
  • kits comprising a magnetic device capable of generating a three-dimensional magnetic field, a microfluidic cartridge device having one or more removable chambers for containing a biological sample, wherein the microfluidic cartridge is configured to be secured with magnetic device such that upon securing with the magnetic device the microfluidic cartridge device is exposed to a three- dimensional magnetic field generated with the pneumatic actuation device, and a binding buffer comprising PEG.
  • kits are not limited to a particular percentage of PEG within the binding buffer. Indeed, in some embodiments, the percentage of PEG within the binding buffer is any percentage that facilitates optimal nucleic acid purification from a biological sample (e.g., where the purification is selective for DNA greater than 1000 base pairs).
  • the percentage of PEG within the binding buffer is between 6% and 4%. In some embodiments, the percentage of PEG within the binding buffer is between 5.14% and 4.18%. In some embodiments, the binding buffer further comprises nuclease free water and NaCl.
  • kits further comprise solid supports comprising super paramagnetic beads coated with carboxylic acid functional groups.
  • kits further comprise a washing buffer comprising PEG.
  • the percentage of PEG within the washing buffer is between 5% and 20%. In some embodiments, the percentage of PEG within the washing buffer is between 7.5% and 16%. In some embodiments the washing buffer further comprises MgCl 2 and NaCl.
  • the micro fluidic cartridge device is secured with the magnetic device.
  • the magnetic device is part of a pneumatic actuation device.
  • kits further comprise a sequencing device (e.g., a next generation sequencing device).
  • a sequencing device e.g., a next generation sequencing device.
  • the technology provides methods for producing a sequencing library from a biological sample, comprising mixing a sample comprising nucleic acid fragments with a binding buffer and solid supports, wherein the mixing results in a binding of the nucleic acid fragments with the solid supports, wherein the mixing occurs within a removable chamber secured with a microfluidic cartridge, wherein the microfluidic cartridge is secured with a magnetic device capable of generating a three-dimensional magnetic field, wherein the solid supports comprise super paramagnetic beads coated with carboxylic acid functional groups, wherein the binding buffer comprises PEG, generating a sequencing library through segregating the bound nucleic acid fragments through exposing the mixed sample to a three-dimensional magnetic field generated with the magnetic device, and delivering the sequencing library to a sequencing device (e.g., a next generation sequencing device).
  • a sequencing device e.g., a next generation sequencing device
  • the percentage of PEG within the binding buffer is any percentage that facilitates optimal nucleic acid purification from a biological sample (e.g., where the purification is selective for DNA greater than 1000 base pairs). In some embodiments, the percentage of PEG within the binding buffer is between 6% and 4%. In some embodiments, the percentage of PEG within the binding buffer is between 5.14% and 4.18%. In some embodiments, the binding buffer further comprises nuclease free water and NaCl.
  • the segregated nucleic acid fragments are primarily greater than 1000 base pairs (e.g., greater than 50% of the segregated nucleic acid fragments are over 1000 bps; greater than 70%> are over 1000 bps; greater than 80%> are over 1000 bps; greater than 90%) are over 1000 bps; greater than 95% are over 1000 bps; etc) (e.g., the average of the segregated nucleic acid fragments are over 1000 bps).
  • the methods further comprise washing the segregated nucleic acid fragments prior to delivering the sequencing library to a sequencing instrument.
  • the washing comprises contacting the segregated nucleic acid fragments with a washing buffer comprising PEG, wherein the percentage of PEG within the washing buffer is between 5% and 20%>.
  • the washing buffer further comprises MgCl 2 and NaCl. In some embodiments, the percentage of PEG within the washing buffer is between 7.5% and 16%.
  • the methods further comprise eluting the segregated nucleic acid fragments.
  • the binding buffer further comprises nuclease free water and
  • the magnetic device is part of a pneumatic actuation device. Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figs. 1-3 show percentage DNA yield recovery as a function of PEG percentage within a binding buffer.
  • Figs. 4-5 show percentage DNA yield recovery as a function of number of base pairs within a wash buffer having varying amounts of ethanol.
  • Fig. 6 shows percentage DNA yield recovery as a function of number of base pairs within a wash buffer having varying percentages of PEG.
  • Fig. 7 shows percentage DNA yield recovery as a function of number of base pairs within a wash buffer having varying percentages of PEG and 20mM MgCl 2 .
  • Fig. 8 shows a comparison of DNA yield recovery through use of a wash buffer having either ethanol or 10% PEG and 20mM MgCl 2 .
  • biological sample is used in a broad sense and is intended to include a variety of biological sources that contain nucleic acids.
  • Such sources include, without limitation, whole tissues, including biopsy materials and aspirates; in vitro cultured cells, including primary and secondary cells, transformed cell lines, and tissue and cellular explants; whole blood, red blood cells, white blood cells, and lymph; body fluids such as urine, sputum, semen, secretions, eye washes and aspirates, lung washes, cerebrospinal fluid, abscess fluid, and aspirates.
  • biological sample include samples processed from biological sources, including but not limited to cell lysates and nucleic acid- containing extracts. Any organism containing nucleic acid may be a source of a biological sample, including, but not limited to, human organisms.
  • isolated nucleic acid refers to the recovery of nucleic acid molecules from a source. While it is not always optimal, the process of recovering nucleic acid may also include recovering some impurities such as protein. It includes, but is not limited to, the physical enrichment of nucleic acid molecules from a source. The term “isolating” may also refer to the duplication or amplification of nucleic acid molecules, without necessarily removing the nucleic acid molecules from the source.
  • nucleic acid refers to a polymer of ribonucleosides or deoxyribonucleosides typically comprising phosphodiester linkages between subunits. Other linkages between subunits include, but are not limited to, methylphosphonate,
  • nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, fragmented nucleic acid, nucleic acid obtained from subcellular organelles such as mitochondria or chloroplasts, and nucleic acid obtained from microorganisms or DNA or RNA viruses that may be present on or in a biological sample.
  • Solid phase components that are capable of binding to nucleic acids released from a biological sample include a variety of materials that are capable of binding nucleic acids under suitable conditions.
  • An example of a solid phase is a super paramagnetic carboxyl bead.
  • Paramagnetic and superparamagnetic materials e.g., when fabricated as microbeads
  • Paramagnetic and superparamagnetic materials have the property of responding to an external magnetic field when present, but dissipating any residual magnetism immediately upon release of the external magnetic field, and are thus easily resuspended and remain monodisperse, but when placed in proximity to a magnetic field, clump tightly, the process being fully reversible by simply removing the magnetic field.
  • magnetic force field refers to a volume defined by the magnetic flux lines between two poles of a magnet or two faces of a coil. Electromagnets and driving circuitry can be used to generate magnetic fields and localized magnetic fields. Permanent magnets may also be used. Preferred permanent magnetic materials include NdFeB
  • Magnetic forces within a magnetic force field follow the lines of magnetic flux. Magnetic forces are strongest where magnetic flux is most dense. Magnetic force fields penetrate most solids and liquids. A moving magnetic force field has two vectors: one in the direction of travel of the field and the other in the direction of the lines of magnetic flux.
  • microfiuidic cartridge refers to a device, cartridge, chip, or card with fluidic structures (e.g., channels, chambers, voids, etc.) having microfiuidic dimensions, e.g., at least one internal cross-sectional dimension that is less than
  • microfiuidic flow regime is characterized by Poiseuille or "laminar" flow (see, e.g., Staben et al. 2005. Particle transport in Poiseuille flow in narrow channels. Intl J Multiphase Flow 31 :529-47, and references cited therein).
  • Microfiuidic devices may be fabricated from various materials using techniques such as laser stenciling, embossing, stamping, injection molding, masking, etching, and three- dimensional soft lithography. Laminated microfiuidic devices are further fabricated with adhesive interlayers or by thermal adhesiveless bonding techniques, such as by pressure treatment of oriented polypropylene. The microarchitecture of laminated and molded microfiuidic devices can differ.
  • the microfiuidic cards of the present technology are designed to interact or "dock" with a host instrument that provides a control interface and optional temperature and magnetic interfaces. The card, however, generally contains all biological reagents needed to perform the assay and requires only application of a sample or samples. These cards are generally disposable, single-use, and are generally manufactured with sanitary features to minimize the risks of exposure to biohazardous material during use and upon disposal.
  • nucleic acid refers to a polymeric form of nucleotides of any length, including but not limited to, ribonucleotides and deoxyribonucleotides. Relatively short (e.g., 10 to 1000 nt or bp) nucleic acid polymers are often used as "primers” or “probes”.
  • the definition encompasses nucleic acids from natural sources, e.g., that are methylated or capped, and synthetic forms, e.g., that contain substitute or derivatized nucleobases and that are based on a peptide backbone.
  • Nucleic acids are generally polymers of adenosine, guanine, thymine, and cytosine and their "deoxy" forms, but may also contain other pyrimidines such as uracil and xanthine or spacers and universal bases such as deoxyinosine. Deoxynucleic acids may be single-stranded or double-stranded depending on the presence or absence of complementary sequences and on conditions of pH, salt concentration, temperature, and the presence or absence of certain organic solvents such as formamide, ⁇ , ⁇ -dimethylformamide, dimethylsulfoxide, and n- methylpyrrolidinone.
  • a sample comprising nucleic acids may include, e.g., one or more metagenomes, genomes, chromosomes, genes, portions of genes, regulatory sequences of genes, mRNAs, rRNAs, tRNAs, siRNAs, miRNAs, cDNA, and may be single stranded, double stranded, or triple stranded.
  • Some nucleic acids have polymorphisms, deletions, and alternate splice sequences.
  • Standard methods for purifying and size selecting DNA sequencing libraries utilizes Solid Phase Reversible Immobilization (SPRI) paramagnetic bead-based technology.
  • SPRI Solid Phase Reversible Immobilization
  • NGS next generation sequencing
  • SPRI beads do not have a suitable magnetic moment for use in automated micro fluidics.
  • size selecting properties are limited such that typically two separate rounds of size selecting purifications are needed to have enough size selection.
  • wash buffers having ethanol which is not compatible with microfluidics and can inhibit downstream applications.
  • the provided compositions, methods and kits address such issues and provide optimized binding buffers and wash buffers suitable for use with automated microfluidic sample prep systems for making NGS libraries.
  • kits used for generating DNA libraries that are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other nucleic acid analysis technologies.
  • methods, compositions, and related kits for size selective nucleic acid purification of next generation sequencing libraries using paramagnetic beads with an optimal magnetic moment, a size selecting binding buffer, and a wash buffer compatible with automated microfluidics are suitable for use in sequencing methods (e.g., next generation sequencing methods) or other nucleic acid analysis technologies.
  • methods, compositions, and related kits for size selective nucleic acid purification of next generation sequencing libraries using paramagnetic beads with an optimal magnetic moment, a size selecting binding buffer, and a wash buffer compatible with automated microfluidics are provided herein.
  • samples e.g., biological samples comprising cells and/or nucleic acids.
  • samples include various fluid samples.
  • the sample is a bodily fluid sample from the subject.
  • the sample is an aqueous or a gaseous sample.
  • the sample is a gel.
  • the sample includes one or more fluid component.
  • the sample comprises tissue collected from a subject.
  • the sample comprises a bodily fluid, secretion, and/or tissue of a subject.
  • the sample is a biological sample.
  • the biological sample is a bodily fluid, a secretion, and/or a tissue sample.
  • biological samples include but are not limited to, blood, serum, saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, breath, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or
  • the sample is provided from a human or an animal, e.g., in some embodiments the sample is provided from a mammal (e.g., a vertebrate) such as a murine, simian, human, farm animal, sport animal, or pet. In some embodiments, the sample is collected from a living subject and in some embodiments the sample is collected from a dead subject.
  • a mammal e.g., a vertebrate
  • the sample is collected from a living subject and in some embodiments the sample is collected from a dead subject.
  • methods for a size selective nucleic acid purification from a biological sample comprise: selectively binding nucleic acid having a desired size to a solid phase by contacting the biological sample with the solid phase under conditions which selectively bind nucleic acid having a desired size; washing the solid phase with the bound nucleic acid with a wash buffer; separating the solid phase with the bound nucleic acid from an unbound portion of the biological sample; and isolating the nucleic acid from the solid phase.
  • the solid phase is a super paramagnetic bead.
  • the super paramagnetic bead is a super paramagnetic bead coated with carboxyl acid functional groups (super paramagnetic carboxyl bead).
  • the step of separating the solid phase with the bound nucleic acid from an unbound portion of the biological sample is a magnetic separation.
  • a magnetic separation is accomplished with a magnetic device capable of generating a three-dimensional magnetic field.
  • the magnetic device capable of generating a three-dimensional magnetic field is part of a pneumatic actuation device.
  • the pneumatic actuation device utilizes multiple electromagnets including a "top magnet” so as to ensure z-axis mixing (e.g., three dimensional mixing).
  • Such methods for size selective nucleic acid purification from a biological sample are not limited to purifying a particular size of the nucleic acid.
  • the methods are selective for purifying nucleic acid greater than approximately 1000 base pairs (e.g., 950 bps, 1000 bps, 1500 bps, 2000 bps, 3000 bps, 5000 bps, 7500 bps, 10000 bps, etc).
  • the conditions which selectively bind such nucleic acid comprise using a binding buffer comprising: polyethylene glycol (PEG) (e.g., PEG 8000) (between 4% and 6% w/v PEG; 4.18% and 5.14% w/v PEG, e.g., 4.8% PEG), sodium chloride, and water
  • a binding buffer wherein the percentage of PEG within the binding buffer is between 4 and 6% is optimal for methods for nucleic acid purification from a biological sample, where the purification is selective for DNA greater than 1000 base pairs (see, Example I).
  • the conditions which selectively bind such nucleic acid comprise using a wash buffer comprising: 10%> Tween 20, PEG (between 5% and 20%> w/v PEG (e.g., PEG 8000), e.g., 7.5% to 16% PEG), sodium chloride, and approximately 20 mM MgCl 2 .
  • a wash buffer wherein the percentage of PEG within the wash buffer is between 5% and 20% is optimal for methods for nucleic acid purification from a biological sample, where the purification is selective for DNA greater than 1000 base pairs (see, Example I).
  • the wash buffer is compatible with optical detection based NGS technologies (e.g., in some embodiments the wash buffer does not produce a fluorescence emission that interferes with NGS technologies).
  • Such methods for size selective nucleic acid purification from a biological sample may be conducted in any desired setting. In certain embodiments, such methods are conducted within a microfluidic device. The methods are not limited to a particular type of microfluidic device.
  • the microfluidic device is a microfluidic cartridge device. In certain embodiments, the microfluidic cartridge device has one or more removable chambers for receiving and containing the biological sample to be purified, the super paramagnetic carboxyl beads, the binding buffer, and the wash buffer.
  • the microfluidic cartridge device is configured to be secured with a magnetic device (e.g., a magnetic device capable of generating a three-dimensional magnetic field) such that upon securing with the magnetic device the microfluidic cartridge device is exposed to a three-dimensional magnetic field generated with the magnetic device.
  • a magnetic device e.g., a magnetic device capable of generating a three-dimensional magnetic field
  • the magnetic device capable of generating a three-dimensional magnetic field is part of a pneumatic actuation device.
  • the microfluidic device is an integrated microfluidics cartridge for preparing libraries for next generation sequencing (NGS).
  • NGS next generation sequencing
  • the microfluidic cartridge accepts an input sample that is a biological sample and provides as output a nucleic acid sequencing library suitable for NGS.
  • experiments conducted during the course of developing such embodiments determined that use of superparamagnetic carboxyl beads, a binding buffer having a percentage of PEG between 4% and 6%, and a wash buffer having a percentage of PEG between 5% and 20% was not only optimal for selecting DNA greater than 1000 base pairs, but also was optimal for use within an automated microfluidic system.
  • Such methods for size selective nucleic acid purification from a biological sample can be used for any number of purposes. In certain embodiments, such methods are used for generating a library of DNA having greater than 1000 base pairs. In some embodiments, such a library is useful for next generation sequencing.
  • such methods are used for preparing nucleic acids for construction of a sequencing library, purifying nucleic acids having a desired size (e.g., greater than 1000 base pairs), and constructing a sequencing library.
  • such methods are integrated into a microfluidic cartridge that contains one or more (e.g., in some embodiments, all) reagents needed to perform the process (e.g., superparamagnetic carboxyl beads, binding buffers (e.g., having a percentage of PEG between 4% and 6%) and wash buffers (e.g., having a percentage of PEG between 5% and 20%)).
  • the cartridges generally contain a port where the resulting output (e.g., a sequencing library) is directly introduced into the workflow of an apparatus that performs DNA sequencing, such as an Illumina sequencing instrument (e.g., a HiSeq, NextSeq, MiSeq, or other instrument based on sequencing-by-synthesis, e.g., Solexa sequencing-by-synthesis), a Life Technologies sequencing instrument (e.g., an Ion Proton, Ion PGM, or other instrument based on the Ion Torrent technology), or a PacBio sequencing instrument (e.g., an RS II, an instrument based on the SMRT technology, or other Pac Bio sequencing
  • an Illumina sequencing instrument e.g., a HiSeq, NextSeq, MiSeq, or other instrument based on sequencing-by-synthesis, e.g., Solexa sequencing-by-synthesis
  • Life Technologies sequencing instrument e.g., an Ion Proton, Ion PGM, or other instrument based on the
  • the technology is suitable for these extant sequencing technologies, platforms, workfiows, and instruments, the technology is not limited to providing sequencing libraries that are compatible with these exemplary technologies. Accordingly, the technology is general and provides sequencing libraries to any existing, nascent, or future sequencing technology. Further, in some embodiments, the resulting output nucleic acids are used and/or processed using other nucleic acid research and/or analysis techniques (e.g., microarray, transformation, solution hybridization, phage display, cloning (e.g., shotgun cloning, construction of clone libraries, construction of metagenomic libraries, etc.).
  • nucleic acid research and/or analysis techniques e.g., microarray, transformation, solution hybridization, phage display, cloning (e.g., shotgun cloning, construction of clone libraries, construction of metagenomic libraries, etc.).
  • a nucleic acid sequencing library is prepared from an input sample comprising whole cells (e.g., eukaryotic, bacteria, archaea), viruses, environmental samples, and/or tissue. Accordingly, in some embodiments, nucleic acids are obtained from the cells or viruses prior to continuing with other various sample preparation operations.
  • the collected cells, viral coat, etc. are treated to prepare a crude extract (e.g., a cell lysate), followed by additional operations (e.g., mixing the sample with superparamagnetic carboxyl beads, binding buffers (e.g., having a percentage of PEG between 4% and 6%), and wash buffers (e.g., having a percentage of PEG between 5% and 20%).
  • a crude extract e.g., a cell lysate
  • additional operations e.g., mixing the sample with superparamagnetic carboxyl beads, binding buffers (e.g., having a percentage of PEG between 4% and 6%), and wash buffers (e.g., having a percentage of PEG between 5% and 20%).
  • desalting of the sample and isolation of the nucleic acid are carried out in a single step, e.g., by binding the nucleic acids to a solid phase (e.g., superparamagnetic carboxyl beads) and washing away the contaminating material.
  • a solid phase e.g., superparamagnetic carboxyl beads
  • the nucleic acid is subjected to one or more preparative reactions (e.g., following sample collection, lysis, and/or nucleic acid extraction).
  • preparative reactions e.g., following sample collection, lysis, and/or nucleic acid extraction.
  • exemplary embodiments are associated with preparative reactions that include in vitro transcription, labeling, fragmentation, amplification, and other reactions.
  • Nucleic acid amplification increases the number of copies of the target nucleic acid sequence of interest.
  • a variety of amplification methods are suitable for use in the methods and device described herein, including, e.g., the polymerase chain reaction (PCR), the ligase chain reaction (LCR), self sustained sequence replication (3SR), and nucleic acid based sequence amplification (NASBA).
  • nucleic acids are labeled, e.g., to facilitate subsequent steps and/or to provide for detection of the nucleic acids.
  • labeling is performed during amplification.
  • amplification incorporates a label into the amplified nucleic acid either through the use of labeled primers or the incorporation of labeled dNTPs into the amplified nucleic acid.
  • the nucleic acids are labeled following amplification.
  • Post amplification labeling typically involves the covalent attachment of a particular detectable group to the amplified nucleic acids.
  • Suitable labels or detectable groups include a variety of fluorescent or radioactive labeling groups well known in the art. These labels are coupled in various embodiments to the sequences using methods that are well known in the art. See, e.g., Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition.
  • the label comprises, but is not limited to, one or more fluorescent labels (including, but not limited to, FITC, PE, Texas RED, Cyber Green, JOE, FAM, HEX, TAMRA, ROX, Alexa 488, Alexa 532, Alexa 546, Alexa 405, or other fluorescent labels (including, but not limited to, FITC, PE, Texas RED, Cyber Green, JOE, FAM, HEX, TAMRA, ROX, Alexa 488, Alexa 532, Alexa 546, Alexa 405, or other fluorescent labels (including, but not limited to, FITC, PE, Texas RED, Cyber Green, JOE, FAM, HEX, TAMRA, ROX, Alexa 488, Alexa 532, Alexa 546, Alexa 405, or other fluorescent labels (including, but not limited to, FITC, PE, Texas RED, Cyber Green, JOE, FAM, HEX, TAMRA, ROX, Alexa 488, Alexa 532, Alexa 546, Alexa 40
  • radioactive labels including, but not limited to, P, H, or 14 C
  • fluorescent proteins including, but not limited to, GFP, RFP, or YFP
  • quantum dots gold particles, sliver particles, biotin, beads (including but not limited magnetic beads or polystyrene beads).
  • embodiments provide that the purified nucleic acids are subjected to other treatments.
  • embodiments are related to fragmenting nucleic acids prior to subsequent steps.
  • Fragmentation of nucleic acids may generally be carried out by physical, chemical, or enzymatic methods that are known in the art. These treatments may be performed within the amplification chamber, or alternatively, may be carried out in a separate chamber (e.g., a nucleic acid fragmentation chamber or in the cell lysis chamber).
  • physical fragmentation methods include but are not limited to moving the sample containing the nucleic acid over pits or spikes in the surface of a reaction chamber or fluid channel.
  • the microfluidic cartridge comprises a component for purifying and size selecting nucleic acids such as, e.g., a collection of nucleic acid fragments or a nucleic acid sequencing library (e.g., for NGS, e.g., an NGS library).
  • the component for purifying and size selecting nucleic acids comprises a chamber to hold a nucleic acid sample.
  • a binding buffer (wherein the percentage of PEG within the binding buffer is between 4% and 6%) and super paramagnetic carboxyl beads (e.g., beads having a high magnetic moment appropriate for use in a microfluidic cartridge and an affinity for nucleic acid) are added to the nucleic acid to bind nucleic acids having a specified range of fragment sizes.
  • the binding buffer is formulated to promote the binding of nucleic acids having a specified range of fragment sizes to the beads.
  • binding buffer that selects for fragments of nucleic acid having a length of at least approximately 1000 base pairs (e.g., to remove small fragments having approximately less than 1000 base pairs).
  • a binding buffer selective for nucleic acid having a length of at least approximately 1000 base pairs comprises polyethylene glycol (PEG) (between 4.18% and 5.14% w/v PEG (e.g., PEG 8000), e.g., 4.8% PEG) sodium chloride, and water (molecular biology grade water, e.g., nuclease free water).
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • water molecular biology grade water, e.g., nuclease free water
  • some embodiments comprise use of a wash buffer to remove the smaller fragments.
  • the wash buffer comprises approximately 10%> Tween-20, PEG (between 5% and 20% w/v PEG (e.g., PEG 8000), e.g., 7.5% to 16% PEG) and approximately 20 mM MgCl 2 .
  • the wash buffer is compatible with optical detection based NGS technologies (e.g., in some embodiments the wash buffer does not produce a fluorescence emission that interferes with NGS technologies).
  • the microfluidic cartridge comprises a component or module for purifying and size selecting nucleic acids.
  • the component or module comprises a chamber to hold a sample and a reservoir comprising paramagnetic beads with a high magnetic moment and a size selecting binding buffer.
  • the component or module comprises a chamber to hold a sample, a reservoir comprising paramagnetic beads with a high magnetic moment, and a reservoir comprising a size selecting binding buffer.
  • the component or module for purifying and size selecting nucleic acids comprises a reservoir comprising a wash buffer that is compatible with microfluidics (e.g., that does not comprise an alcohol, e.g., ethanol or isopropanol, for instance).
  • the reservoirs comprising the beads, binding buffer, and wash buffer are in fluid communication with the chamber for containing the sample.
  • the chamber for containing the sample is in fluid communication with one or more other components or modules that provide the sample to the component or module for purifying and size selecting nucleic acids and/or that receive the size selected sample from the component or module for purifying and size selecting nucleic acids.
  • the microfluidic cartridge comprises a component or module (e.g., a reaction chamber) for ligating adaptors to nucleic acids.
  • a component or module e.g., a reaction chamber
  • the term “adaptor” encompasses both adaptors (e.g., “linkers”) that comprise single stranded nucleotide overhangs at the 5' and/or 3' ends or that do not comprise single stranded nucleotide overhangs at the 5' and/or 3' ends, e.g., a blunt ended adaptor.
  • one or more single stranded overhangs comprise 1, 2, or more nucleotides.
  • adaptors comprise additional nucleic acid sequence for cloning or for the analysis of "inserts.”
  • adaptors comprise labels or affinity tags for analysis or purification of "inserts.”
  • Ligating an adaptor to a nucleic acid fragment is performed using a ligation reaction, which is a biochemical reaction in which an enzyme (e.g., a ligase) forms a chemical link between a nucleic acid fragment and an adaptor.
  • a ligation reaction which is a biochemical reaction in which an enzyme (e.g., a ligase) forms a chemical link between a nucleic acid fragment and an adaptor.
  • Ligation methods are known in the art and utilize standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition). Such methods utilize ligase enzymes such as DNA ligase to effect or catalyze joining of the ends of two polynucleotide strands by forming a covalent linkage.
  • an adaptor comprises a 5 '-phosphate moiety to facilitate ligation to the nucleic acid fragment 3'-OH.
  • a nucleic acid fragment comprises a 5'- phosphate moiety, either residually from the shearing process, or added using an enzymatic treatment step.
  • a nucleic acid fragment has been end repaired and, optionally, extended to produce an overhanging base or bases, to give a 3'-OH suitable for ligation.
  • joining means covalent linkage of polynucleotide strands which were not previously covalently linked.
  • joining comprises formation of a phosphodiester linkage between the two polynucleotide strands, but other means of covalent linkage (e.g. non-phosphodiester backbone linkages) may be used.
  • Many ligation methods utilize either a blunt or TA-mediated ligation.
  • the ligase is a T4 ligase, a variant of T4 DNA ligase, an E. coli ligase, etc.
  • an oligonucleotide adapter is ligated onto a nucleic acid fragment as a step to prepare a sequencing library for sequencing (e.g., NGS).
  • a sequencing library for sequencing e.g., NGS
  • an end polishing step is performed on nucleic acid fragments to create blunt ends on the nucleic acid fragments.
  • an enzymatic reaction e.g., a PCR, terminal transferase reaction, or Klenow exo minus polymerase reaction adds an untemplated A to the ends of nucleic acids.
  • some embodiments of ligation reactions comprise ligating adaptors with blunt ends and some embodiments of ligation reactions comprise ligating adaptors with overhangs, e.g., with T overhangs that are complementary to the A overhang on the nucleic acid.
  • the microfluidic cartridge receives a sample (e.g., an aqueous biological sample), extracts nucleic acids from the sample, amplifies the nucleic acids, prepares the amplified nucleic acids for sequencing (e.g., by fragmenting the nucleic acid, optionally polishing the ends of the nucleic acid fragments, and ligating adaptors to the nucleic acid fragments to provide a NGS library), and delivers the NGS library directly to an NGS workflow, instrument, and/or sequencer for sequencing.
  • a sample e.g., an aqueous biological sample
  • amplifies the nucleic acids e.g., by fragmenting the nucleic acid, optionally polishing the ends of the nucleic acid fragments, and ligating adaptors to the nucleic acid fragments to provide a NGS library
  • sequencing e.g., by fragmenting the nucleic acid, optionally polishing the ends of the nucleic acid fragments, and ligating adaptors to the
  • the microfluidic cartridge produces an NGS library for NGS sequencing and delivers the NGS library to the NGS sequencer without the sample being touched or transported by a person, e.g., which reduces error, contamination, and other problems associated with the transport of samples by a person.
  • nucleic acid library e.g., a nucleic acid library that is produced as an output of a microfluidic cartridge as described herein.
  • the present technology is not limited by the type of sequencing method employed. Exemplary sequencing methods are described below.
  • nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.
  • 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. 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.
  • next-generation sequencing is a set of methods referred to as "next-generation sequencing” techniques.
  • Most current methods describe the use of next-generation sequencing technology for de novo sequencing of whole genomes to determine the primary nucleic acid sequence of an organism.
  • targeted re- sequencing deep sequencing allows for sensitive mutation detection within a population of wild-type sequence.
  • NGS technology produces large amounts of sequencing data points.
  • a typical run can easily generate tens to hundreds of megabases per run, with a potential daily output reaching into the gigabase range. This translates to several orders of magnitude greater than a standard 96-well plate, which can generate several hundred data points in a typical multiplex run.
  • Target amplicons that differ by as little as one nucleotide can easily be distinguished, even when multiple targets from related species are present. This greatly enhances the ability to do accurate genotyping.
  • NGS alignment software programs used to produce consensus sequences can easily identify novel point mutations, which could result in new strains with associated drug resistance.
  • the use of primer bar coding also allows multiplexing of different patient samples within a single sequencing run.
  • NGS methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods. NGS methods can be broadly divided into those that require template amplification and those that do not. Amplification-requiring methods include pyrosequencing developed by Solexa and commercialized by Illumina. Non-amplification approaches, also known as single-molecule sequencing, include the Ion Torrent platform commercialized by Life Technologies and the molecule real time sequencing (also known as SMRT) technologies developed by Pacific Biosciences.
  • 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.
  • A-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.
  • 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.
  • reaction volume approximately 20 zepto liters (20 x 10 liters).
  • 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.
  • single molecule real time (SMRT) DNA sequencing methods using zero-mode waveguides (ZMWs) developed by Pacific Biosciences, or similar methods are employed. With this technology, DNA sequencing is performed on SMRT chips, each containing thousands of zero-mode waveguides (ZMWs).
  • ZMWs zero-mode waveguides
  • a ZMW is a hole, tens of nanometers in diameter, fabricated in a 100 nm metal film deposited on a silicon dioxide substrate.
  • Each ZMW becomes a nanophotonic visualization chamber providing a detection volume of just 20 zepto liters (10 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.
  • Phospholinked 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.
  • each base is held within the detection volume for tens of milliseconds, which is orders of magnitude longer than the amount of time it takes a nucleotide to diffuse in and out of the detection volume.
  • the engaged fluorophore emits fluorescent light whose color corresponds to the base identity.
  • the polymerase cleaves the bond holding the fluorophore in place and the dye diffuses out of the detection volume.
  • the signal immediately returns to baseline and the process repeats.
  • Unhampered and uninterrupted, the DNA polymerase continues incorporating bases at a speed of tens per second. In this way, a completely natural long chain of DNA is produced in minutes. Simultaneous and continuous detection occurs across all of the thousands of ZMWs on the SMRT chip in real time.
  • PacBio have demonstrated this approach has the capability to produce reads thousands of nucleotides in length.
  • nanopore sequencing is employed (see, e.g., Astier et al, Am
  • 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.
  • the nanopore may be a solid-state pore fabricated on a metal and/or nonmetal surface, or a protein-based nanopore, such as alpha-hemolysin (Clarke et al., Nat. Nanotech., 4, Feb. 22, 2009: 265-270).
  • 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.
  • Other single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al, Clinical Chem., 55: 641-658, 2009; U.S. Pat. No.
  • Some embodiments of the technology are related to systems for processing a biological sample (e.g., a sample comprising cells and/or nucleic acid).
  • a biological sample e.g., a sample comprising cells and/or nucleic acid.
  • embodiments provide systems for producing a sequencing library for input into an NGS workflow.
  • embodiments of the technology provide a system for production of a NGS library from a biological sample, the system comprising two or more of a microfluidic cartridge to accept as input a biological sample and produce a NGS sequencing library as output, superparamagnetic carboxyl beads, a binding buffer (e.g., having a percentage of PEG between 4% and 6%), a wash buffer (e.g., having a percentage of PEG between 5% and 20%), and a magnetic device capable of generating a three-dimensional magnetic field.
  • the system comprises a piezoelectric component and a thermoelectric component.
  • the system comprises a power supply to provide a voltage and current to the system components.
  • the system comprises a frequency regulator.
  • Some embodiments provide systems for sequencing a nucleic acid (e.g., using NGS). For example, some embodiments provide a system comprising an apparatus for producing an NGS library for input to an NGS workflow and/or an NGS sequencer.
  • the methods and systems described herein are associated with a programmable machine designed to perform a sequence of arithmetic or logical operations as provided by the methods described herein.
  • some embodiments of the technology are associated with (e.g., implemented in) computer software and/or computer hardware.
  • the technology relates to a computer comprising a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a processor or a microprocessor) for executing a series of instructions (e.g., a method as provided herein) to read, manipulate, and store data.
  • a processor comprises one or more processors.
  • a processor provides instructions to one or more valves, components, modules, thermoelectric components, piezoelectric components, pumps, reagent supplies, etc. in the microfluidic cartridge and/or apparatus.
  • a microprocessor is part of a system comprising one or more of a CPU, a graphics card, a user interface (e.g., comprising an output device such as a display and an input device such as a keyboard), a storage medium, and memory components.
  • Memory components e.g., volatile and/or nonvolatile memory find use in storing
  • Programmable machines associated with the technology comprise conventional extant technologies and technologies in development or yet to be developed (e.g., a quantum computer, a chemical computer, a DNA computer, an optical computer, a spintronics based computer, etc.).
  • Some embodiments provide a computer that includes a computer-readable medium.
  • the embodiment includes a random access memory (RAM) coupled to a processor.
  • the processor executes computer-executable program instructions stored in memory.
  • processors may include a microprocessor, an ASIC, a state machine, or other processor, and can be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California and Motorola Corporation of Schaumburg, Illinois.
  • Such processors include, or may be in communication with, media, for example computer-readable media, which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.
  • Embodiments of computer-readable media include, but are not limited to, an electronic, optical, magnetic, or other storage or transmission device capable of providing a processor, such as the processor of client, with computer-readable instructions.
  • suitable media include, but are not limited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM, RAM, an ASIC, a configured processor, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read instructions.
  • various other forms of computer-readable media may transmit or carry instructions to a computer, including a router, private or public network, or other transmission device or channel, both wired and wireless.
  • the instructions may comprise code from any suitable computer-programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, Swift, Ruby, Unix, and JavaScript.
  • Computers are connected in some embodiments to a network or, in some
  • Computers may also include a number of external or internal devices such as a mouse, a CD-ROM, DVD, a keyboard, a display, or other input or output devices.
  • Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smart phones, pagers, digital tablets, laptop computers, internet appliances, and other processor-based devices.
  • the computer-related to aspects of the technology provided herein may be any type of processor-based platform that operates on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, etc., capable of supporting one or more programs comprising the technology provided herein. All such components, computers, and systems described herein as associated with the technology may be logical or virtual.
  • sequencing data are produced. Following the production of sequencing data, the sequencing data are reported to a data analysis operation in some embodiments. To facilitate data analysis in some embodiments, the sequencing data are analyzed by a digital computer. In some embodiments, the computer is appropriately programmed for receipt and storage of the sequencing data and for analysis and reporting of the sequencing data gathered, e.g., to provide a nucleic acid sequence in a human or machine readable format.
  • Example I This example demonstrated the optimization of a size selecting binding buffer a size selecting wash buffer.
  • Binding buffers consists of Polyethylene Glycol (PEG), Sodium Chloride, and Nuclease Free Water.
  • the formulations are designed to size select for a particular fragment range. A goal was to find a binding buffer formulation that would size select for pieces >1000 base pairs (eliminating smaller libraries inserts). At the same time, the recovery of nucleic acids had to be efficient enough so that there was sufficient material available for sequencing.
  • a 2 log DNA size ladder (New England Biolabs) that consists of DNA fragments that range in size from 0.1 to 10 kb was used. The protocol for each sample is listed in Table 1.
  • binding buffer T10/15/20 binding buffer T10/15/20.
  • PEG percentages less than 4.18 yielded too low of a recovery to have sufficient DNA for sequencing.
  • Fig. 6 shows percentage yield recovery with a wash buffer having 7.5% PEG, 1.4M NaCI or 70% ethanol).
  • adding magnesium chloride (20mM MgCl 2 ) to the wash buffer and increasing the PEG (from 7.5% to 16%) of the wash buffer increased the percentage yield (Fig. 7).
  • a wash buffer having 10% PEG, 1.4M NaCl and 20mM MgCl 2 resulted in the most optimal percentage recovery. Indeed, the percentage recovery is almost double over use of a wash buffer having 70% ethanol (Fig. 8).

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Abstract

La présente invention concerne des procédés, des compositions et des kits utilisés pour la génération de bibliothèques d'ADN qui sont appropriées pour être utilisées dans des procédés de séquençage (par exemple, des procédés de séquençage de nouvelle génération) ou d'autres technologies d'analyse d'acide nucléique. En particulier, la présente invention concerne des procédés, des compositions et des kits associés pour la purification par taille de bibliothèques de séquençage de nouvelle génération en utilisant des billes paramagnétiques avec un moment magnétique optimal, un tampon de liaison de sélection de taille, et un tampon de lavage compatible avec les microfluides.
PCT/US2015/057182 2014-10-24 2015-10-23 Systèmes, compositions et procédés pour la purification d'acide nucléique par taille WO2016065298A1 (fr)

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