WO2025178954A1 - Procédés de séquençage et de comptage d'acides nucléiques - Google Patents

Procédés de séquençage et de comptage d'acides nucléiques

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Publication number
WO2025178954A1
WO2025178954A1 PCT/US2025/016475 US2025016475W WO2025178954A1 WO 2025178954 A1 WO2025178954 A1 WO 2025178954A1 US 2025016475 W US2025016475 W US 2025016475W WO 2025178954 A1 WO2025178954 A1 WO 2025178954A1
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WO
WIPO (PCT)
Prior art keywords
sequence
sample
random
reads
sequencing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/016475
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English (en)
Inventor
Robert MELTZER
Christopher D'amato
Yi XUE
Trinity SMITHERS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Illumina Inc
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Illumina Inc
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Filing date
Publication date
Application filed by Illumina Inc filed Critical Illumina Inc
Publication of WO2025178954A1 publication Critical patent/WO2025178954A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • 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 invention provides methods for nucleic acid sequencing in which the number of sequence reads is representative of the number of molecules that were present in a sample.
  • nucleic acids in a sample are copied in a manner such that each copy has a segment of bases that uniquely identifies the original molecule. This can be done by capturing the target molecules with primer oligonucleotides that each have a random sequence at the 3' end. The primer oligonucleotides anneal to the target molecules at random locations, due the random 3' ends. When the primer oligonucleotides are extended to copy the target molecules, the 5' ends of the resultant copies will include random stretches of bases.
  • the random start sites provide each of the copies of the nucleic acid molecules with a unique sequence.
  • the unique sequence in each of the copies is copied (directly or as, e.g., a second-strand copy) from a segment of a transcript transcribed by an organism.
  • aspects of the disclosure provide methods of preparing a sample for sequencing and counting molecules therein.
  • the methods include providing a sample comprising a plurality of sample polynucleotides and generating a plurality of copies of the sample polynucleotides.
  • a copy of the plurality of copies comprises (i) a sample sequence copied from a sample polynucleotide of the plurality of sample polynucleotides, and (ii) a tag sequence copied from the sample polynucleotide, the tag sequence distinguishing said sample polynucleotide from other sample polynucleotides from the sample.
  • Dual IMI embodiments may include fragmenting the plurality of sample polynucleotides at a plurality of random break sites. Each copy may include the tag sequence copied from the sample polynucleotide at a 5' end and a second tag sequence at a 3' end, the second tag sequence copied from the sample polynucleotide from a segment adjacent one break site of the plurality of random break sites.
  • FIG. 2 shows compositions used in methods of the invention.
  • FIG. 3 shows melting temperatures over [Mg++] for randomer capture sequences.
  • the invention provides methods and compositions that are useful for nucleic acid sequencing methods.
  • claimed methods are useful with particle-templated instant partitions (PIPs) when sequencing target nucleic acids (PIPseq).
  • PIPs particle-templated instant partitions
  • Methods disclosed herein are optimized for preserved, fixed, or otherwise challenging samples such as formalin-fixed, paraffin embedded (FFPE) tissue samples.
  • Methods and compositions of the invention are quantitative in that they are useful to determine identities and quantities of individual molecules present in samples.
  • methods herein are useful for transcriptomics, to measure and show what quantities of different RNA transcripts are being expressed in samples.
  • aqueous partitions such as PIPs, methods may be performed for individual cells and even for multiple individual cells that are liberated from, or interrogated within, a single sample.
  • a count of reads that map to each transcript provides a count of how many transcripts from each gene are in a sample (e.g., single cell) and those counts can serve as expression levels, or an expression profile, or a transcriptome of the sample.
  • An IMI may be understood to be a transcript-informed sequence that is also used for transcript capture using a randomer capture sequence or that is copied from a random fragmentation position. With random priming (or random fragmentation) the identifying information at the end(s) of each molecule is generated by the transcript sequence. That identifying information is a function of gene identity and the position at which each transcript was annealed to the capture moiety. Randomer capture with IMI analysis has multiple potential uses including, for example, analysis of non-polyadenylated RNA; bacterial transcriptomics; viral gene transcription; analysis of non-coding RNA (IncRNA, etc.); analysis on sequencing platforms that only support single-end reads; and sequencing on other platforms such as Ultima sequencing.
  • Methods may use an IMI or may use dual-IMIs.
  • dual IMI embodiments each molecule has randomized initiation positions at both 3’ and 5’ ends.
  • Dual IMI analysis may have particular advantages in identification of very short transcript fragments.
  • a specific application of dual-IMI approaches may include recovery of severely fragmented RNA from clinical formaldehyde fixed samples or forensic or archeological samples.
  • Preferred embodiments of the disclosure subject the target nucleic acid to fragmentation, even to fragmentation conditions and chemistries that would be recognized as too promiscuous for conventional methods, causing too many random breaks, yielding many short fragments that terminate at essentially random points of breakage.
  • the 3' random ends of the capture oligos anneal to those fragments. After a capture oligo anneals to a fragment, it is extended by a polymerase or reverse transcriptase — copying the fragment until the enzyme reaches the end, the random point of breakage, of the fragment.
  • the enzyme generates a copy of the fragment for which the 5' end starts with the capture oligo and the 3' includes a copy of the broken end of the fragment (plus any additional sequence that may be added 3' of that copy).
  • Some embodiments use template-switching oligos.
  • Some embodiments use blunt-ending and adapter ligation.
  • the 5' end of the copy has one IMI generated by random sequence of the capture oligo.
  • the 3' end of the copy has a second IMI generated by copying the segment of the fragment near the random point of breakage.
  • each copy of one of the fragments includes first and second IMIs, or dual IMIs.
  • a transposase is used to randomly cut the cDNA.
  • Tn5 transposase
  • RNA sequencing by direct tagmentation of RNA/DNA hybrids PNAS117 (6) 2886- 2893, incorporated by reference.
  • the Tn5 transposase randomly binds and cuts doublestranded RNA/DNA and attaches its end sequence to the random cut site.
  • some embodiments of the invention use Tn5 transposase to directly tagment RNA/DNA hybrids and form polynucleotide libraries with intrinsic molecular identifiers (essentially unique sequences of bases originating in genetic material of the organism or biological system being studied).
  • Tn5 a RNase H superfamily member
  • the desired oligo is preferably a PCR handle (aka a universal primer binding site, a sequencing adaptor, a synthetic oligo of known sequence to which a PCR primer anneals, etc.).
  • Methods of the invention may be used with various amounts of input sample, from single cells to large numbers of cells, with a dynamic range spanning numerous orders of magnitude.
  • each copy of a fragment has a first IMI at one end and a second IMI at the other end.
  • Those copies are preferably subject to amplification (either with adapter ligation or with tailed primers) to generate amplicons (e.g., with sequencing adaptors and/or primer binding sites at both ends).
  • amplicons e.g., with sequencing adaptors and/or primer binding sites at both ends.
  • all of the amplicons have first and second IMIs at their respective ends.
  • an IMI functions like a molecular barcode, it is not the case that dual IMIs are equivalent to a single, but double-length, barcode.
  • Methods and compositions of the invention are compatible with cell preparation techniques that include aldehyde or formaldehyde fixation protocols.
  • Cell fixation and cell preservation techniques incorporate programmable fixation times, reversible bond formation and cleavage, chemo-selective reactions, and analyte recovery using, e.g., materials and techniques such as those discussed in Gallion, 2021, Preserving single cells in space and time for analytical assays, Trends Anal Chem 122: 115723, incorporated by reference.
  • Samples may be obtained from fixed cells or fixed tissue blocks. Samples that are formaldehyde-fixed, paraffin-embedded (FFPE) may be used.
  • FFPE formaldehyde-fixed, paraffin-embedded
  • methods of the disclosure may include steps from nuclei extraction from FFPE and formaldehyde fixed tissue. According to methods of the invention, fixed cells or nuclei may be captured as normal. Methods may include treatment with proteinase K, optionally using heat-triggered proteinase K activation, with added lysis buffer enzymes to efficiently dissolve and liberate cells and/or nuclei.
  • nucleic acids of interest typically include mRNA or precursor mRNA transcripts.
  • Other RNAs, such as ribosomal RNA (rRNA) may not be of-interest to the assay being performed.
  • Methods of the invention include a step for the depletion or removal of nontarget nucleic acids such as rRNA.
  • Ribosomal RNA removal using CRISPR may generally be referred to as CRISPR-based depletion or CRISPR-depletion or CRISPR ribodepletion or similar when applied to ribosomal RNA.
  • CRISPR-depletion technology harnesses the specificity of CRISPR to degrade abundant, uninformative sequences.
  • CRISPR-depletion may be integrated into a stranded total RNA sequencing library prep protocol by adding Cas9/RNA complexes after the adapter ligation step.
  • the Cas9/RNA complexes include a pool of guide RNAs that target and deplete unwanted sequences.
  • CRISPR-depletion technologies are not specific to sequencing platforms.
  • CRISPR-depletion or bulk ribodepletion reagents may be used to remove overabundant human, mouse, or rat rRNA from RNA-Seq libraries to improve sequencing sensitivity and performance. Kits are available commercially to provide a bulk post-library depletion reagent with multiplexing format - designed to remove overabundant human 5S, 5.8S, 18S, 28S, 45S (precursor), mitochondrial 12S and 16S ribosomal RNA.
  • CRISPR-based depletion may include the methods and materials for CRISPR-depletion sold under the trademark CRISPRCLEAN by Jumpcode Genomics, Inc. (San Diego, CA).
  • CRISPR-depletion may use materials or techniques described in US 2022/0145359 or US 2023/0265528, incorporated by reference.
  • magnetic pull-down depletion is an option, using rRNA-specific probes linked to magnetic beads, or example.
  • Single cell embodiments may involve isolating single cells into aqueous partitions or compartments to sequence and count nucleic acid molecules of a single cell. Isolation into partitions may be accomplished by any suitable mechanism and any suitable type of aqueous partition may be used. Exemplary suitable partitions include droplets, wells in a plate, or other fluid portioning structures. For example, the partitions may be wells, cavities, pockets, or openings in a pico-, nano-, or microtiter plate or substrate, or fluidic harbors (see, e.g., US Pub 2010/0041046 Al, incorporated by reference).
  • the partitions may be well in a multi-well plate such as a 96-well plate, 384 well plate, a 1536 well plate, a 3456 well plate, or a 9600 well plate.
  • the partitions may be separate chambers (see, e.g., 20210178395 Al, incorporated by reference).
  • the partitions may be distinct regions defined within a fluidic device (see, e.g., 20200269248 Al, incorporated by reference).
  • the partitions are droplets of an emulsion such as a water-in-oil (W/O) emulsion or a water-in-oil-in-water (W/O/W) emulsion.
  • W/O water-in-oil
  • W/O/W water-in-oil-in-water
  • template particles are in the mixture that is sheared or vortexed (e.g., dozens, hundreds, thousands, tens of thousands, more) is the number of droplets that are formed (as well as some "satellite droplets, or mere bubbles, of miscellaneous sizes and integrities that are not relevant to downstream analysis and can be simply ignored).
  • the resultant droplets that each include a template particle are monodisperse (e.g., same number of polymer subunits among the beads aka particles, and/or same mass each, and/or essentially same size/volume among the particles, e.g., same diameter when viewed under a microscope)
  • the resultant droplets that each include a template particle are monodisperse (each the size of one template particle plus a small, thin shell of aqueous fluid around it). If a droplet forms that contains two or more template particles, then during shearing or vortexing, that droplet breaks into one droplet per each template particle.
  • the capture moiety e.g., 3' sequence of the hybrid capture oligo
  • genomic information that is encoded within the sequence of the source gene for a molecule. Where the other end is generated by random fragmentation, both ends of each molecule are informed by the specific gene identity and the position of the molecule along that gene.
  • FIG. 2 shows compositions used in methods of the invention.
  • Methods and compositions of the invention preferably use a template particle 205, such as a hydrogel bead, linked to one or more capture oligos 207.
  • a 3' end of the capture oligo 207 defines a primer 219 for hybrid capture of a target nucleic acid 213.
  • the primer 219 preferably includes a stretch of random bases and may be referred to as a randomer or random priming sequence. Any suitable number (e g., from 3 to 30, or fewer, or more) bases may constitute the random priming sequence.
  • Preferred embodiments use about 6 to about 10, e.g., 8, random bases to make up the primer 219.
  • the capture oligo hybridizes to the target nucleic acid 213 at a random location and primes the synthesis of a first strand copy 235, where a 5' end of the first strand copy 235 begins at a random start location 232 in the target nucleic acid 231.
  • the first strand copy has a 3' end that includes a copy of bases adjacent a second random location 231.
  • Amplification may be performed to generate amplicons ready for sequencing, or sequencing libraries, from the target molecules.
  • Each amplicon may have an IMI or dual IMIs.
  • the disclosed system may use IMIs to deduplicate sequence reads and count template molecules in the sample.
  • Preferred embodiments use dual IMIs.
  • the dual IMI system involves randomer capture (by a segment of random bases on a 3' end of the capture oligo, e.g., that is attached to a bead) to read a random sequence from a 3 1 end of each molecule (e.g., template RNA).
  • Libraries have randomized segments at both 5' and 3' ends of molecules. Either or both may be used as IMI(s) for sequence read deduplication.
  • Attachment of the end sequence to the cDNA at the random cut site produces a construct, a contiguous DNA molecule that includes a first PCR handle (PEI), a cell barcode, a capture segment, a portion of the cDNA optionally terminating at the random cut site, and a second PCR handle (PE2).
  • PEI first PCR handle
  • PE2 cell barcode
  • Amplification of the construct yields amplicons.
  • constructs are amplified with a P5-PE1 hybrid oligo and P7 index primer directly into a sequencing library.
  • the library may be sequenced to assess RNA expression, for example, as described in Hrdlickova, 2017, RNA-Seq methods for transcriptome analysis, WIREs RNA 8(1): 10.1002, incorporated by reference.
  • Constructs or amplicons may include certain primer and index sequences or copies thereof, such as, P5s and P7s.
  • Those sequences may be any arbitrary sequence useful in downstream analysis. For example, they may be additional universal primer binding sites or sequencing adaptors.
  • either or both of the P5s and P7s may be arbitrary universal priming sequence (universal meaning that the sequence information is not specific to the naturally occurring genomic sequence being studied but is instead suited to being amplified using a pair of cognate universal primers, by design).
  • the index segment may be any suitable barcode or index such as may be useful in downstream information processing.
  • Libraries may be sequenced by any suitable method. Suitable methods include Sanger sequencing, Illumina or Ultima sequencing, Roche pyrosequencing, single-molecule, long-read sequencing using platforms offered by Pacific Biosciences or Oxford Nanopore.
  • An example of a sequencing technology that can be used is Illumina sequencing. Illumina sequencing is based on the amplification of DNA on a solid surface using fold-back PCR and anchored primers. Genomic DNA is fragmented and attached to the surface of flow cell channels. Four fluorophore-labeled, reversibly terminating nucleotides are used to perform sequencing. After nucleotide incorporation, a laser is used to excite the fluorophores, and an image is captured, and the identity of the first base is recorded.
  • SAM sequence alignment map format
  • Other methods useful for processing and analyzing sequence reads are discussed in U.S. Pat. No. 8,209,130, which is incorporated by reference. Determining gene expression generally involves counting numbers of unique sequence reads that uniquely map to a human reference genome. Mapping reads to a reference to identify genes may be performed using computer software packages known in the art.
  • mapping reads to a reference and identifying genes gives a quantitative result when reads are deduplicated to yield one read per mRNA from which those reads originated. Because each mRNA is typically copied into cDNA and each cDNA is typically copied into an unpredictably large number of amplicons in the sequencing library, and because each library member is often amplified or read redundantly as part of a sequencing technique, a number of raw sequence reads does not necessarily correlate to numbers of input molecules from the single cells. Nevertheless, one cell may include abundant transcripts that map to one gene.
  • compositions and methods of the invention give each cDNA at least one unique intrinsic identifier (and preferably dual-IMIs) that can be identified within, and used to deduplicate, sequence reads. After those sequence reads are identified by gene and deduplicated, then counts of those reads provide a quantitative measure of gene expression levels. Methods may include, prior to the deduplicating step, saving the sequence reads in memory, coupled to at least on processor in a computer system, as a FASTA or FASTQ file, wherein the deduplicating and mapping are performed by the computer system. [0069] As discussed, methods of the invention are useful for scRNA-Seq and specifically for expression analysis.
  • cells are isolated into, and lysed within, aqueous partitions with capture oligos.
  • the capture oligos anneal to RNAs released from the cells.
  • the capture oligos preferably include partition-specific barcodes and PCR handles. Once the capture oligos have hybridized to the RNAs, those duplexes may be released from partitions and pooled at any subsequent stage. Because capture oligos with partition-specific barcodes are used to capture and tag RNA from cells isolated in the partition, any arbitrary number of cells may be captured in parallel (simultaneously).
  • the cell barcodes in the sequencing data can be used to “bin” the sequence data by original cell, i.e., assign each sequence read (or assembled contigs or sequences therefrom) back to originating cells.
  • Read deduplication 131 and transcript counting is preferably performed by a computer system operably linked to a sequencing instrument and executing program instructions causing the computer system to perform those functions.
  • sequence reads may arrive at the computer system in FASTQ format.
  • Each entry — each "sequence read — in a FASTQ file (or FASTA) will include a segment of sequence information read from the target molecules.
  • Those sequence reads will also each include at least one IMI.
  • each target molecule will generate two reads, a forward read and a reverse read, and each read will have an IMI read from the target molecule.
  • sequence information is read from the target molecule is used to identify the gene of origin for that molecule (e.g., transcript). But each IMI is also read from the target molecule.
  • the computer system can execute program software to deduplicate the reads (simply identifying all duplicates and saving only one, i.e., "collapsing" the reads to a single read; or by leaving the FASTQ files intact but only "counting" all duplicate reads as 1, e.g., in a count file.
  • the system may also identify gene information for each read, e.g., by mapping to a reference or by querying a transcript database.
  • Read mapping can proceed by known methods including, for example, by methods that involve pairwise alignment of each read to a reference, such as a published human genome such as the 36 th build of the human genome refer e d to in industry as HG36 or hg36. Comparison to references may also proceed by building hashes of k-mers in the reference and in the query (the sequence read) and looking up the hashed k-mers of the query in the target (the reference). Read-mapping may involve transforming each sequence in order or in characters via an informatic transform such as the Burroughs-Wheeler transform (BWT) after which comparison of the BWT of the query to the target is trivial.
  • BWT Burroughs-Wheeler transform
  • RNA transcript a gene (as it is found in the DNA within the genome of an organism that is being studied) that has been transcribed into an RNA transcript, that was copied with a randomer and amplified to generate amplicons with at least one IMI, which amplicons are sequenced to generate sequence reads that include the IMI is assigned to the sequence read, and by implication, the RNA transcript is identified as having been transcribed from that gene. For that gene, each unduplicated read is used to increment a count of transcripts by one.
  • the read counts are a measure of a number of actual transcript molecules that were present in the sample.
  • the gene identities and read counts are a measure of expression levels of the gene in that cell and are also thus a transcriptome or transcriptomic profile for the cell.
  • Each copy includes (i) a sample sequence copied from a sample polynucleotide of the plurality of sample polynucleotides, and (ii) a tag sequence copied from the sample polynucleotide, the tag sequence distinguishing said sample polynucleotide from other sample polynucleotides from the sample.
  • the tag sequence functions like an IMI discussed above.
  • the information of the tag sequence is information that was in the sample polynucleotides originally, i.e., it is genetic information of the organism being studied.
  • Generating the copies may include annealing, to the sample polynucleotide, a primer (e.g., random er of a capture oligo) having a random sequence of bases at a 3' end and extending the primer to generate the tag sequence.
  • the random sequence may be about 8 bases in length, e.g., about 6 to 9.
  • the plurality of sample polynucleotides may be RNA and each of the plurality of copies of the sample polynucleotides may include a random tag sequence copied from the RNA.
  • the unique sequence in each of the copies is copied from a segment of a transcript transcribed by an organism.
  • the methods may include performing the recited steps for a plurality of cells to generate, for each cell of the plurality, a transcriptome profile based on, for that cell, the count of mapped unique reads.
  • the fluid may comprise reagents such as surfactants (e.g., octylphenol ethoxylate and/or octylphenoxypolyethoxyethanol), reducing agents (e.g., DTT, beta mercaptoethanol, or combinations thereof).
  • surfactants e.g., octylphenol ethoxylate and/or octylphenoxypolyethoxyethanol
  • reducing agents e.g., DTT, beta mercaptoethanol, or combinations thereof.

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Abstract

L'invention concerne des procédés de séquençage (127) et de comptage d'acides nucléiques. Les procédés peuvent comprendre la réalisation de copies (113) de molécules d'acide nucléique à partir de sites de départ aléatoires, le séquençage (127) des copies pour obtenir des lectures de séquence, la déduplication (131) des lectures de séquence pour produire uniquement des lectures uniques, et le mappage des lectures uniques sur des gènes et, pour chacun des gènes, la fourniture d'un nombre de lectures uniques mappées.
PCT/US2025/016475 2024-02-23 2025-02-19 Procédés de séquençage et de comptage d'acides nucléiques Pending WO2025178954A1 (fr)

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