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US 12,529,099 B2Granted
Nucleotide sequence generation by barcode bead- colocalization in partitions
AssigneeBio-Rad Laboratories, Inc.
InventorsRonald Lebofsky, Andrew Kohlway, Jennifer Chew, Zachary Burkett, Man Cheng
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Abstract
Comparison of common sequencing reads from sequencing based on partition-based barcoding can be used to improve sequencing results. Increased loading of barcodes per partition can also improve sequencing results.
Claims13
A method of generating a nucleotide sequence of a DNA template from DNA from a cell, the method comprising:
- (a) contacting the DNA with a transposase that cleaves DNA in the DNA sample to form DNA fragments and introduces adaptor sequences to ends of the DNA fragments, to form adaptor-modified DNA fragments;
- (b) partitioning the adaptor-modified DNA fragments into a plurality of partitions, the partitions comprising a particle comprising a solid support surface, the solid support surface having a plurality of oligonucleotide primers conjugated thereon, wherein the oligonucleotide primers comprise a barcode sequence and wherein different particles are distinguished by having different barcodes from one another, wherein at least some partitions have more than one particles per partition, and wherein different particles are conjugated to primers having different barcode sequences;
- (c) in the partitions, amplifying the adaptor-modified DNA fragments to form adaptor-modified DNA fragment amplicons and linking oligonucleotide primers from the solid support to the adaptor-modified DNA fragment amplicons, thereby forming barcoded adaptor-modified DNA fragment amplicons;
- (d) combining barcoded adaptor-modified DNA fragment amplicons from multiple partitions;
- (e) generating a plurality of sequencing reads of the barcoded adaptor-modified DNA fragment amplicons;
- (f) determining in a pairwise manner percent of sequencing reads of identical DNA fragments sequences shared between different barcodes;
- (g) comparing the determined percent of identical DNA fragment sequences shared between different barcodes to a threshold value, wherein if two barcodes have a determined percent of identical DNA fragment sequences in common above the threshold value, the two barcodes are determined to be in the same partition;
- and
- (h) generating a nucleotide sequence for the DNA template from the plurality of sequencing reads, wherein generating the nucleotide sequence comprises treating sequencing reads having different barcodes determined to be in the same partition as being from the same partition;
- thereby generating a nucleotide sequence of a DNA template.
7 more elements
A method of distinguishing differently barcoded sequence reads originating from different partitions from sequence reads having different barcodes but originating from the same partition, the method comprising:
- (a) contacting DNA from a cell with a transposase that cleaves DNA in the DNA sample to form DNA fragments and introduces adaptor sequences to ends of the DNA fragments, to form adaptor-modified DNA fragments;
- (b) partitioning the adaptor-modified DNA fragments into a plurality of partitions, the partitions comprising a particle comprising a solid support surface, the solid support surface having a plurality of oligonucleotide primers conjugated thereon, wherein the oligonucleotide primers comprise a barcode sequence and wherein different particles are distinguished by having different barcodes from one another, wherein at least some partitions have more than one particles per partition, and wherein different particles are conjugated to primers having different barcode sequences;
- (c) in the partitions, amplifying the adaptor-modified DNA fragments to form adaptor-modified DNA fragment amplicons and linking oligonucleotide primers from the solid support to the adaptor-modified DNA fragment amplicons, thereby forming barcoded adaptor-modified DNA fragment amplicons;
- (d) combining barcoded adaptor-modified DNA fragment amplicons from multiple partitions;
- (e) generating a plurality of sequencing reads of the barcoded adaptor-modified DNA fragment amplicons;
- (f) determining in a pairwise manner percent of sequencing reads of DNA fragments shared between different barcodes;
- and
- (g) comparing the determined percent of DNA fragments shared between different barcodes to a threshold value, wherein if two barcodes have a determined percent of identical DNA fragments in common above the threshold value, the two barcodes are determined to be in the same partition.
5 more elements
Dependencies are inferred from the claim text.