Methods and systems for processing polynucleotides

a polynucleotide and polynucleotide technology, applied in the field of methods and systems for processing polynucleotides, can solve the problems of many challenges that remain unaddressed or relatively poorly addressed by the solutions currently being offered, and achieve the effect of facilitating the hybridization of the poly-t sequen

Active Publication Date: 2019-08-29
10X GENOMICS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0010]In some embodiments, the nucleic acids are released from the individual cell in the discrete partition. In some embodiments, the nucleic acids comprise ribonucleic acid (RNA), such as, for example, messenger RNA (mRNA). In some embodiments, generating one or more first nucleic acid sequences includes subjecting the nucleic acids to reverse transcription under conditions that yield the one or more first nucleic acid sequences. In some embodiments, the reverse transcription occurs in the discrete partition. In some embodiments, the oligonucleotides are provided in the discrete partition and include a poly-T sequence. In some embodiments, the reverse transcription comprises hybridizing the poly-T sequence to at least a portion of each of the nucleic acids and extending the poly-T sequence in template directed fashion. In some embodiments, the oligonucleotides include an anchoring sequence that facilitates hybridization of the poly-T sequence. In some embodiments, the oligonucleotides include a random priming sequence that can be, for example, a random hexamer. In some embodiments, the reverse transcription comprises hybridizing the random priming sequence to at least a portion of each of the nucleic acids and extending the random priming sequence in template directed fashion.

Problems solved by technology

Despite these advances in biological characterization, many challenges still remain unaddressed, or relatively poorly addressed by the solutions currently being offered.

Method used

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  • Methods and systems for processing polynucleotides
  • Methods and systems for processing polynucleotides
  • Methods and systems for processing polynucleotides

Examples

Experimental program
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example i

Cellular RNA Analysis Using Emulsions

[0173]In an example, reverse transcription with template switching and cDNA amplification (via PCR) is performed in emulsion droplets with operations as shown in FIG. 9A. The reaction mixture that is partitioned for reverse transcription and cDNA amplification (via PCR) includes 1,000 cells or 10,000 cells or 10 ng of RNA, beads bearing barcoded oligonucleotides / 0.2% Tx-100 / 5× Kapa buffer, 2× Kapa HS HiFi Ready Mix, 4 μM switch oligo, and Smartscribe. Where cells are present, the mixture is partitioned such that a majority or all of the droplets comprise a single cell and single bead. The cells are lysed while the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of mRNA that is released from the cell as in operation 950. The poly-T segment is extended in a reverse transcription reaction as in operation 952 and the cDNA transcript is amplified as in operation...

example ii

Cellular RNA Analysis Using Emulsions

[0175]In another example, reverse transcription with template switching and cDNA amplification (via PCR) is performed in emulsion droplets with operations as shown in FIG. 9A. The reaction mixture that is partitioned for reverse transcription and cDNA amplification (via PCR) includes Jurkat cells, beads bearing barcoded oligonucleotides / 0.2% TritonX-100 / 5× Kapa buffer, 2× Kapa HS HiFi Ready Mix, 4 μM switch oligo, and Smartscribe. The mixture is partitioned such that a majority or all of the droplets comprise a single cell and single bead. The cells are lysed while the barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of mRNA that is released from the cell as in operation 950. The poly-T segment is extended in a reverse transcription reaction as in operation 952 and the cDNA transcript is amplified as in operation 954. The thermal cycling conditions are 42° C...

example iii

RNA Analysis Using Emulsions

[0176]In another example, reverse transcription is performed in emulsion droplets and cDNA amplification is performed in bulk in a manner similar to that as shown in FIG. 9C. The reaction mixture that is partitioned for reverse transcription includes beads bearing barcoded oligonucleotides, 10 ng Jurkat RNA (e.g., Jurkat mRNA), 5× First-Strand buffer, and Smartscribe. The barcoded oligonucleotides are released from the bead, and the poly-T segment of the barcoded oligonucleotide hybridizes to the poly-A tail of the RNA as in operation 961. The poly-T segment is extended in a reverse transcription reaction as in operation 963. The thermal cycling conditions for reverse transcription are one cycle at 42° C. for 2 hours and one cycle at 70° C. for 10 min. Following thermal cycling, the emulsion is broken and RNA and cDNA transcripts are denatured as in operation 962. A second strand is then synthesized by primer extension with a primer having a biotin tag as...

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Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 16 / 052,431, filed Aug. 1, 2018, which is a continuation-in-part of U.S. application Ser. No. 16 / 000,803, filed Jun. 5, 2018, which is a continuation of U.S. application Ser. No. 15 / 850,241, filed Dec. 21, 2017, which is a continuation of U.S. patent application Ser. No. 15 / 588,519, filed May 5, 2017, now U.S. Pat. No. 9,856,530, which is a continuation of U.S. patent application Ser. No. 15 / 376,582, filed Dec. 12, 2016, now U.S. Pat. No. 9,701,998, which is a continuation-in-part of U.S. patent application Ser. No. 14 / 104,650, filed on Dec. 12, 2013, now U.S. Pat. No. 9,567,631, which claims priority to U.S. Provisional Application No. 61 / 737,374, filed on Dec. 14, 2012; U.S. patent application Ser. No. 15 / 376,582 is also a continuation-in-part of U.S. patent application Ser. No. 14 / 250,701, filed on Apr. 11, 2014, which is a continuation of U.S. patent application Ser. No. 1...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/6874C12Q1/6869C12Q1/6806C12Q1/6855
CPCC12Q1/6874C12Q1/6869C12Q1/6806C12Q1/6855C12Q2563/149C12Q2563/159C12Q2563/179C12Q2565/519C12Q2521/301C12Q2525/185C12Q2527/125C12Q2531/10C12Q2535/122C12Q2565/629
Inventor HINDSON, BENJAMINHINDSON, CHRISTOPHERSCHNALL-LEVIN, MICHAELNESS, KEVINJAROSZ, MIRNASAXONOV, SERGEHARDENBOL, PAULBHARADWAJ, RAJIVZHENG, XINYINGBELGRADER, PHILLIP
Owner 10X GENOMICS
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