Analysis of nucleic acid sequences

Patent metadata

Field	Value
Publication number	US-2021123103-A1
Application number	US-202016898984-A
Country	US
Kind code	A1
Filing date	Jun 11, 2020
Priority date	Jun 26, 2014
Publication date	Apr 29, 2021
Grant date	—

Abstract

The present disclosure relates to methods, compositions and systems for haplotype phasing and copy number variation assays. Included within this disclosure are methods and systems for combining the barcode comprising beads with samples in multiple separate partitions, as well as methods of processing, sequencing and analyzing barcoded samples.

First claim

1 .- 54 . (canceled)

55 . A method of determining a presence of a structural variation of a nucleic acid, comprising:
(a) providing a plurality of first fragment molecules of the nucleic acid, wherein a given first fragment molecule of the plurality of first fragment molecules comprises the structural variation; (b) sequencing a plurality of second fragment molecules of each of the plurality of first fragment molecules to provide a plurality of fragment sequences, wherein each of the plurality of fragment sequences corresponding to a given first fragment molecule shares a common barcode sequence; and (c) determining the presence of the structural variation by (i) mapping the plurality of fragment sequences to a reference sequence, (ii) identifying the plurality of fragment sequences that share the common barcode sequence, and (iii) identifying the structural variation based on a presence of an elevated amount of the plurality of fragment sequences sharing the common barcode sequence that map to the reference sequence at locations that are further apart than a length of the given first fragment molecule, which elevated amount is relative to a sequence lacking the structural variation.

56 . The method of claim 55 , wherein the elevated amount is 1% or more with respect to a total number of the first fragment molecules that are derived from a region of the nucleic acid having the structural variation.

57 . (canceled)

58 . The method of claim 55 , wherein the locations are at least about 100 bases apart.

59 .- 61 . (canceled)

62 . The method of claim 55 , further comprising identifying the structural variation by creating an assembly of the given first fragment molecule from the plurality of fragment sequences, wherein the plurality of fragment sequences are selected as inputs for the assembly based upon a presence of the common barcode sequence.

63 . The method of claim 62 , wherein the assembly is created by generating a consensus sequence from the plurality of fragment sequences.

64 . The method of claim 55 , wherein the structural variation comprises a translocation.

65 .- 73 . (canceled)

74 . A method of identifying variants in a sequence of a nucleic acid, comprising:
obtaining nucleic acid sequences of a plurality of individual fragment molecules of the nucleic acid, the nucleic acid sequences of the plurality of individual fragment molecules each having a length of at least 1 kilobase (kb); linking sequences of one or more of the plurality of individual fragment molecules in one or more inferred contigs; and identifying one or more variants from the one or more inferred contigs.

75 . (canceled)

76 . The method of claim 74 , wherein the obtaining comprises:
providing a plurality of barcoded fragments of each individual fragment molecule of the plurality of individual fragment molecules, the barcoded fragments of a given individual fragment molecule having a common barcode; sequencing the plurality of barcoded fragments of the plurality of individual fragment molecules, the sequencing providing a sequencing error rate of less than 1%; and determining a sequence of the plurality of individual fragment molecules from sequences of the plurality of barcoded fragments and their associated barcodes.

77 . The method of claim 76 , wherein the linking comprises identifying one or more overlapping sequences between two or more individual fragment molecules to link the two or more individual fragment molecules into the one or more inferred contigs.

78 . The method of claim 77 , wherein the linking comprises identifying one or more common variants between two or more individual fragment molecules to link the two or more individual fragment molecules into the one or more inferred contigs.

79 .- 92 . (canceled)

93 . A method, comprising:
a) partitioning a first nucleic acid into a first partition, where the first nucleic acid comprises the target sequence derived from a first chromosome of an organism; b) partitioning a second nucleic acid into a second partition, where the second nucleic acid comprises the target sequence derived from a second chromosome of the organism; c) in the first partition, attaching a first barcode sequence to fragments of the first nucleic acid or to copies of portions of the first nucleic acid to provide first barcoded fragments; d) in the second partition, attaching a second barcode sequence to fragments of the second nucleic acid or to copies of portions of the second nucleic acid to provide second barcoded fragments, the second barcode sequence being different from the first barcode sequence; e) determining the nucleic acid sequence of the first and second barcoded fragments, and assembling a nucleic acid sequence of the first and second nucleic acids; and f) comparing the nucleic acid sequence of the first and second nucleic acids to characterize the first and second nucleic acids as deriving from first and second chromosomes, respectively.

94 . The method of claim 93 , wherein oligonucleotides comprising the first barcode sequence are co-partitioned with the first nucleic acid, and oligonucleotides comprising the second barcode sequence are co-partitioned with the second nucleic acid.

95 . The method of claim 94 , wherein the oligonucleotides comprising the first barcode sequence are releasably attached to a first bead, and the oligonucleotides comprising the second barcode sequence are releasably attached to a second bead, and the co-partitioning comprises co-partitioning the first and second beads into the first and second partitions, respectively.

96 . The method of claim 93 , wherein the first and second partitions comprise droplets in an emulsion.

97 . 162 . (canceled)

Assignees

• 10X Genomics Inc

Inventors

• Schnall-Levin Michael
• Jarosz Mirna
• Hindson Christopher
• Ness Kevin
• Saxonov Serge
• Hindson Benjamin
• Zheng Xinying
• Marks Patrick
• Stuelpnagel John

Classifications

• C12Q2535/122

  Massive parallel sequencing · CPC title

• G16B30/20

  Sequence assembly · CPC title

• G16B30/10

  Sequence alignment; Homology search · CPC title

• C12Q1/6827 Primary

  for detection of mutation or polymorphism · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title