Methods and systems for barcoding nucleic acid molecules from individual cells or cell populations

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.

What is claimed: 
     
         1 . A method for nucleic acid sequencing, comprising:
 (a) providing a plurality of droplets, wherein a droplet of said plurality of droplets comprises (i) a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, and (ii) a bead comprising a nucleic acid barcode molecule coupled thereto, wherein said nucleic acid barcode molecule comprises a barcode sequence;   (b) using said RNA molecule and said nucleic acid barcode molecule to generate a barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, a sequence corresponding to said nucleic acid sequence of said RNA molecule and a complement of said barcode sequence; and   (c) sequencing said barcoded nucleic acid molecule or a derivative thereof.   
     
     
         2 . The method of  claim 1 , wherein said RNA molecule is from a cell. 
     
     
         3 . The method of  claim 2 , wherein said droplet comprises said cell. 
     
     
         4 . The method of  claim 3 , further comprising releasing said RNA molecule from said cell prior to (b). 
     
     
         5 . The method of  claim 1 , wherein said bead comprises a plurality of nucleic acid molecules coupled thereto, wherein said plurality of nucleic acid molecules comprises said nucleic acid barcode molecule. 
     
     
         6 . The method of  claim 5 , wherein each of said plurality of nucleic acid molecules comprises said barcode sequence. 
     
     
         7 . The method of  claim 6 , wherein each of said plurality of nucleic acid molecules comprises an additional barcode sequence that varies across said plurality of nucleic acid molecules. 
     
     
         8 . The method of  claim 1 , wherein said nucleic acid barcode molecule comprises a template switching sequence. 
     
     
         9 . The method of  claim 1 , further comprising, prior to (c), subjecting said barcoded nucleic acid molecule or derivative thereof to nucleic acid amplification. 
     
     
         10 . The method of  claim 9 , wherein said nucleic acid amplification is performed subsequent to releasing said barcoded nucleic acid molecule or derivative thereof from said droplet. 
     
     
         11 . The method of  claim 9 , wherein said nucleic acid amplification is polymerase chain reaction. 
     
     
         12 . The method of  claim 1 , wherein said RNA molecule is a messenger ribonucleic acid (mRNA) molecule. 
     
     
         13 . The method of  claim 1 , wherein in (a) said droplet comprises (i) an additional nucleic acid molecule comprising an additional nucleic acid sequence, and (ii) an additional nucleic acid barcode molecule comprising an additional barcode sequence, and wherein in (b) said additional nucleic acid molecule and said additional nucleic acid barcode molecule are used to generate an additional barcoded nucleic acid molecule comprising, from a 5′ end to a 3′ end, said additional barcode sequence and an additional sequence corresponding to said additional nucleic acid sequence. 
     
     
         14 . The method of  claim 13 , wherein said additional nucleic acid barcode molecule is coupled to said bead. 
     
     
         15 . The method of  claim 1 , wherein (b) comprises extending a primer hybridized to a region at a 3′ end of the RNA molecule in a primer extension reaction, said nucleic acid barcode molecule acting as a template switching oligonucleotide in said primer extension reaction, thereby generating said barcoded nucleic acid molecule comprising, from the 5′ end to the 3′ end, the sequence corresponding to the nucleic acid sequence of the RNA molecule and the complement of the barcode sequence. 
     
     
         16 . The method of  claim 1 , wherein (b) is performed in said droplet. 
     
     
         17 . The method of  claim 15 , further comprising releasing said barcoded nucleic acid molecule or a derivative thereof from said droplet. 
     
     
         18 . The method of  claim 1 , wherein said barcoded nucleic acid molecule further comprises, towards a 5′ end, a functional sequence for permitting said barcoded nucleic acid molecule or a derivative thereof to couple to a flow cell of a sequencer. 
     
     
         19 . The method of  claim 1 , wherein said sequence is a reverse complement of said nucleic acid sequence. 
     
     
         20 . The method of  claim 1 , further comprising, prior to (c), using said barcoded nucleic acid molecule or a derivative thereof and a pair of primers to generate nucleic acid molecules having a target nucleic acid sequence. 
     
     
         21 . The method of  claim 20 , wherein said target nucleic acid sequence comprises a T cell receptor variable region sequence, a B cell receptor variable region sequence, or an immunoglobulin variable region sequence. 
     
     
         22 . The method of  claim 20 , wherein at least one of said pair of primers hybridizes to a constant region of a T cell receptor nucleic acid sequence, a constant region of a B cell receptor nucleic acid sequence, or a constant region of an immunoglobulin nucleic acid sequence. 
     
     
         23 . The method of  claim 20 , wherein said nucleic acid molecules having said target nucleic acid sequence or derivatives thereof are sequenced in (c). 
     
     
         24 . The method of  claim 1 , further comprising releasing said nucleic acid barcode molecule from said bead. 
     
     
         25 . The method of  claim 24 , wherein said nucleic acid barcode molecule is released from said bead before said barcoded nucleic acid molecule is generated. 
     
     
         26 . The method of  claim 24 , wherein said nucleic acid barcode molecule is released from said bead while said barcoded nucleic acid molecule is generated. 
     
     
         27 . The method of  claim 24 , wherein said nucleic acid barcode molecule is released from said bead after said barcoded nucleic acid molecule is generated. 
     
     
         28 . The method of  claim 1 , wherein said bead is a gel bead. 
     
     
         29 . The method of  claim 1 , wherein said barcode sequence is a combinatorial assembly of a plurality of barcode segments. 
     
     
         30 . The method of  claim 29 , wherein plurality of barcode segments comprises at least three segments.