Methods and systems for processing polynucleotides

Abstract

The present disclosure provides methods, compositions and systems for analyzing individual cells or cell populations through a partitioned analysis of contents of individual cells or cell populations, such as cancer cells and cells of the immune system. Individual cells or cell populations may be co-partitioned with processing reagents for accessing cellular contents, and for uniquely identifying the content of a given cell or cell population, and subsequently analyzing the content of the cell and characterizing it as having derived from an individual cell or cell population, including analysis and characterization of nucleic acid(s) from the cell through sequencing.

1 . A method for nucleic acid sequencing, comprising:
 (a) providing a plurality of partitions, wherein a partition of said plurality of partitions comprises:
 (i) a ribonucleic acid (RNA) molecule from a cell comprising a nucleic acid sequence corresponding to an immune receptor gene of the genome of said cell, 
 (ii) a nucleic acid barcode molecule comprising a barcode sequence and a template switching sequence, and 
 (iii) a primer comprising a 3′ sequence that hybridizes to the RNA molecule; 
   (b) in the partition, generating a barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, a sequence complementary to said nucleic acid sequence of said RNA molecule and a complement of said barcode sequence by:
 (i) hybridizing said primer to a region at a 3′ end of said RNA molecule, 
 (ii) extending said primer, using a reverse transcriptase having terminal transferase activity, to generate a nucleic acid product comprising the sequence complementary to the nucleic acid sequence of the RNA molecule and a 3′ end complementary to the template switching sequence of said nucleic acid barcode molecule, 
 (iii) hybridizing said nucleic acid product to said nucleic acid barcode molecule via the template switching sequence, and 
 (iv) extending the nucleic acid product using the nucleic acid barcode molecule as a template; 
   (c) from a portion of a pooled plurality of barcoded nucleic acid molecules comprising the barcoded nucleic acid molecule, amplifying said nucleic acid sequence corresponding to said immune receptor gene to provide an enriched subset of barcoded nucleic acid molecules or derivatives thereof; and   (d) sequencing said enriched subset of barcoded nucleic acid molecules or derivatives thereof and an unenriched subset of barcoded nucleic acid molecules from said pooled plurality of barcoded nucleic acid molecules.   
     
     
         2 . The method of  claim 1 , wherein the nucleic acid barcode molecule is coupled to a particle. 
     
     
         3 . The method of  claim 2 , wherein the particle is a gel bead or a magnetic bead. 
     
     
         4 . The method of  claim 2 , wherein the nucleic acid barcode molecule is released from the particle upon application of a stimulus. 
     
     
         5 . The method of  claim 4 , further comprising releasing the nucleic acid barcode molecule from the particle prior to the (b) generating the barcoded nucleic acid molecule. 
     
     
         6 . The method of  claim 4 , further comprising releasing the nucleic acid barcode molecule from the particle during the (b) generating the barcoded nucleic acid molecule. 
     
     
         7 . The method of  claim 4 , further comprising releasing the nucleic acid barcode molecule from the particle after the (b) generating the barcoded nucleic acid molecule. 
     
     
         8 . The method of  claim 1 , wherein the sequence complementary to the nucleic acid sequence of the RNA molecule of the generated barcoded nucleic acid molecule comprises: a T cell receptor (TCR) variable region sequence, B cell receptor (BCR) variable region sequence, or Immunoglobulin (Ig) variable region sequence. 
     
     
         9 . The method of  claim 1 , wherein the nucleic acid barcode molecule further comprises a unique molecule identifier (UMI) sequence. 
     
     
         10 . The method of  claim 1 , wherein the nucleic acid barcode molecule further comprises a sequencing primer sequence. 
     
     
         11 . The method of  claim 9 , wherein the nucleic acid barcode molecule further comprises a sequencing primer sequence. 
     
     
         12 . The method of  claim 1 , wherein the 3′ sequence of the primer that hybridizes to the RNA molecule comprises a polyT sequence. 
     
     
         13 . The method of  claim 1 , wherein the partition comprises the cell. 
     
     
         14 . The method of  claim 13 , further comprising lysing the cell to obtain the RNA molecule. 
     
     
         15 . The method of  claim 1 , wherein the partition comprises a droplet, microwell or well. 
     
     
         16 . The method of  claim 1 , wherein the 3′ end complementary to the template switching sequence of the nucleic acid barcode molecule comprises a polyC sequence. 
     
     
         17 . The method of  claim 16 , wherein the polyC sequence is added as the 3′ end complementary to the template switching sequence of the nucleic acid barcode molecule via the terminal transferase activity of the reverse transcriptase. 
     
     
         18 . The method of  claim 17 , wherein the template switching sequence comprises a polyG sequence. 
     
     
         19 . The method of  claim 1 , wherein the primer is not attached to a particle. 
     
     
         20 . A method for nucleic acid sequencing, comprising:
 (a) providing a plurality of partitions, wherein a partition of said plurality of partitions comprises:
 (i) a ribonucleic acid (RNA) molecule from a cell comprising a nucleic acid sequence corresponding to an immune receptor gene of the genome of said cell, 
 (ii) a nucleic acid barcode molecule coupled to a particle, wherein the nucleic acid barcode molecule comprises a barcode sequence and a template switching sequence, and 
 (iii) a primer comprising a 3′ sequence that hybridizes to the RNA molecule, wherein the primer is not coupled to the particle; 
   (b) in the partition, generating a barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, a sequence complementary to said nucleic acid sequence of said RNA molecule and a complement of said barcode sequence by:
 (i) hybridizing said primer to a region at a 3′ end of said RNA molecule, 
 (ii) extending said primer, using a reverse transcriptase comprising terminal transferase activity, to generate a nucleic acid product comprising the sequence complementary to the nucleic acid sequence of the RNA molecule and a 3′ end complementary to the template switching sequence of said nucleic acid barcode molecule, 
 (iii) hybridizing said nucleic acid product to said nucleic acid barcode molecule via the template switching sequence, and 
 (iv) extending the nucleic acid product using the nucleic acid barcode molecule as a template; and 
   (c) sequencing the barcoded nucleic acid molecule.   
     
     
         21 . The method of  claim 20 , further comprising pooling the barcoded nucleic acid molecule generated in the partition with other barcoded nucleic acid molecules generated in other partitions to provide a pooled plurality of barcoded nucleic acid molecules. 
     
     
         22 . The method of  claim 21  wherein, from a portion of the pooled plurality of barcoded nucleic acid molecules, amplifying the nucleic acid sequence corresponding to said immune receptor gene to provide an enriched subset of barcoded nucleic acid molecules or derivatives thereof, wherein the barcoded nucleic acid molecule is sequenced from the enriched subset. 
     
     
         23 . The method of  claim 22 , further comprising sequencing an additional barcoded nucleic acid molecule, wherein the additional barcoded nucleic acid molecule is not of the enriched subset. 
     
     
         24 . The method of  claim 20 , wherein the immune receptor gene comprises a T cell receptor (TCR) variable region sequence, a B cell receptor (BCR) variable region sequence, or an Immunoglobulin (Ig) variable region sequence. 
     
     
         25 . The method of  claim 20 , wherein the partition comprises the cell. 
     
     
         26 . The method of  claim 25 , further comprising lysing the cell to obtain the RNA molecule. 
     
     
         27 . The method of  claim 20 , further comprising: amplifying the sequence complementary to the nucleic acid sequence of the RNA molecule of the generated barcoded nucleic acid molecule. 
     
     
         28 . The method of  claim 27 , wherein the sequence complementary to the nucleic acid sequence of the RNA molecule of the generated barcoded nucleic acid molecule comprises: a TCR, BCR, or Ig variable region sequence. 
     
     
         29 . A method, comprising:
 (a) providing a reaction volume comprising
 (i) a cell or cell derivative, and 
 (ii) a bead comprising a barcoded oligonucleotide releasably coupled thereto, wherein said barcoded oligonucleotide is a template switching oligonucleotide; and 
   (b) releasing said barcoded oligonucleotide from said bead to provide said barcoded oligonucleotide in said reaction volume; and   (c) subjecting said reaction volume to an amplification reaction to generate an amplification product, wherein during said amplification reaction, said reaction volume comprises a template polynucleotide from said cell or cell derivative, said barcoded oligonucleotide and a primer having a sequence towards a 3′ end that hybridizes to said template polynucleotide, and wherein said amplification product has sequence complementarity with said template polynucleotide and said barcoded oligonucleotide.   
     
     
         30 . A non-transitory computer-readable medium comprising machine-executable code that, upon execution by one of more computer processors, implements a method for generating a labeled polynucleotide, the method comprising:
 (a) subjecting a reaction mixture to a first reaction under conditions sufficient to generate a first nucleic acid product, wherein said reaction mixture comprises (i) a template polynucleotide, (ii) a primer having a sequence towards a 3∝ end that hybridizes to said template polynucleotide, and (iii) a template switching oligonucleotide, wherein said first nucleic acid product comprises said primer, a reverse complement of a sequence of said template polynucleotide, and a sequence complementary to at least a portion of said template switch oligonucleotide; and   (b) subjecting said first nucleic acid product to a second reaction in the presence of a barcoded oligonucleotide under conditions sufficient to generate a second nucleic acid product, wherein said barcoded oligonucleotide comprises a sequence of at least a segment of said template switching oligonucleotide, wherein (i) said second reaction uses said first nucleic acid as a template and said barcoded oligonucleotide as a primer, or (ii) said second reaction uses said barcoded oligonucleotide as a template and at least a portion of said first nucleic acid as a primer, to generate said second nucleic acid product,   
       wherein said first reaction and said second reaction are performed within a same reaction volume.