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US 10,975,371 B2Granted
Nucleic acid sequence analysis from single cells
AssigneeIllumina, Inc.
InventorsNeeraj Salathia, Jian-Bing Fan, Fiona Kaper, Gordon M. Cann, Arash Jamshidi, Alex Aravanis
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Abstract
Presented herein are methods and compositions for multiplexed single cell gene expression analysis. Some methods and compositions include the use of droplets and/or beads bearing unique barcodes such as unique molecular barcodes (UMI).
Claims38
A method of preparing a cDNA library from a plurality of single cells comprising:
- releasing mRNA from each single cell to provide a plurality of individual mRNA samples, wherein the mRNA in each individual mRNA sample is from a single cell;
- synthesizing a first strand of cDNA from the mRNA in each individual mRNA sample with a first strand synthesis primer mixture, wherein the first strand synthesis primer mixture comprises a mixture of an oligo dT primer and a randomer primer, wherein the oligo dT primer and the randomer primer each comprise a tag to incorporate the tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNA sample is complementary to mRNA from a single cell, and wherein each tag of each tagged cDNA sample has a same first portion comprising a cell-specific identifier sequence, a same first-read sequencing adapter sequence, and a different second portion;
- pooling the tagged cDNA samples generated from a plurality of single cells;
- amplifying the pooled, tagged cDNA samples to generate pooled, tagged, double-stranded cDNA;
- and
- performing a tagmentation reaction on the pooled, tagged, double-stranded cDNA to generate the cDNA library by contacting the pooled, tagged, double-stranded cDNA with a plurality of transposome complexes each comprising a transposase complexed with a transposon comprising a transposon end sequence and a second-read sequencing adapter sequence, wherein the second-read sequencing adapter sequence is different from the first-read sequencing adapter sequence and is identical in all the transposons in the plurality of transposome complexes, wherein the transposome complexes used in the tagmentation reaction do not comprise the first-read sequencing adapter sequence, and wherein the cDNA library comprises a plurality of tagged cDNA fragments having the first-read sequencing adapter sequence on a first strand and the second-read sequencing adapter sequence on a second strand.
3 more elements
A method of preparing a cDNA library from a plurality of single cells comprising:
- releasing mRNA from each single cell to provide a plurality of individual mRNA samples, wherein the mRNA in each individual mRNA sample is from a single cell;
- synthesizing a first strand of cDNA from the mRNA in each individual mRNA sample with a first strand synthesis primer and incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNA sample is complementary to mRNA from a single cell, and wherein the tag comprises a cell-specific identifier sequence, a first-read sequencing adapter sequence, and a unique molecular identifier (UMI) sequence;
- pooling the tagged cDNA samples generated from a plurality of single cells;
- amplifying the pooled cDNA samples to generate pooled, tagged, double-stranded cDNA;
- and
- performing a tagmentation reaction on the pooled, tagged, double-stranded cDNA to generate the cDNA library by contacting the pooled, tagged, double-stranded cDNA with a plurality of transposome complexes each comprising a transposase complexed with a transposon comprising a transposon end sequence and a second-read sequencing adapter sequence that is identical in all the transposons in the plurality of transposome complexes, wherein the second-read sequencing adapter sequence is different from the first-read sequencing adapter sequence, wherein the transposome complexes used in the tagmentation reaction do not comprise the first-read sequencing adapter sequence, and wherein the cDNA library comprises a plurality of tagged cDNA fragments having the first-read sequencing adapter sequence on a first strand and the second-read sequencing adapter sequence on a second strand.
3 more elements
A method of preparing a cDNA library from a plurality of single cells comprising:
- releasing mRNA from each single cell to provide a plurality of individual mRNA samples, wherein the mRNA in each individual mRNA sample is from a single cell;
- synthesizing a first strand of cDNA from the mRNA in each individual mRNA sample with a first strand synthesis primer and incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNA sample is complementary to mRNA from a single cell, and wherein the tag comprises a cell-specific identifier sequence and a unique molecular identifier (UMI) sequence, and wherein the first strand synthesis primer comprises:
- (i) a mosaic end region having paired ends and positioned 5′ of the UMI sequence, and
- (ii) a first-read sequencing adapter sequence in a hairpin region formed between the paired ends of the mosaic end region;
- pooling the tagged cDNA samples generated from a plurality of single cells;
- amplifying the pooled cDNA samples to generate pooled, tagged, double-stranded cDNA;
- and
- performing a tagmentation reaction on the pooled, tagged, double-stranded cDNA to generate the cDNA library by contacting the pooled, tagged, double-stranded cDNA with a plurality of transposome complexes each comprising a transposase complexed with a transposon comprising a transposon end sequence and a second-read sequencing adapter sequence that is identical in all the transposons in the plurality of transposome complexes, wherein the second-read sequencing adapter sequence is different from the first-read sequencing adapter sequence, wherein the transposome complexes used in the tagmentation reaction do not comprise the first-read sequencing adapter sequence, and wherein the cDNA library comprises a plurality of tagged cDNA fragments having the first-read sequencing adapter sequence on a first strand and the second-read sequencing adapter sequence on a second strand.
5 more elements
Dependencies are inferred from the claim text.