Methods and systems for processing polynucleotides

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

1 - 30 . (canceled) 
     
     
         31 . A method for processing a ribonucleic acid (RNA) molecule, comprising:
 (a) providing a partition, which partition comprises (i) a cell comprising said RNA molecule and (ii) a nucleic acid barcode molecule;   (b) in said partition, fragmenting said RNA molecule into a plurality of fragmented RNA molecules; and   (c) using said nucleic acid barcode molecule and a fragmented RNA molecule of said plurality of fragmented RNA molecules, or derivative thereof, to perform a nucleic acid reaction to generate a barcoded RNA molecule.   
     
     
         32 . The method of  claim 31 , wherein said partition is among a plurality of partitions. 
     
     
         33 . The method of  claim 32 , wherein said nucleic acid barcode molecule is different than other nucleic acid barcode molecules in other partitions of said plurality of partitions. 
     
     
         34 . The method of  claim 31 , wherein said partition is a droplet. 
     
     
         35 . The method of  claim 31 , wherein (a) comprises co-partitioning said cell and said nucleic acid barcode molecule to provide said partition. 
     
     
         36 . The method of  claim 31 , wherein said partition is a well. 
     
     
         37 . The method of  claim 31 , where said partition further comprises a bead comprising said nucleic acid barcode molecule coupled thereto. 
     
     
         38 . The method of  claim 37 , wherein said bead is a gel bead. 
     
     
         39 . The method of  claim 31 , wherein (b) comprises using a restriction enzyme to fragment said RNA molecule into said plurality of fragmented RNA molecules. 
     
     
         40 . The method of  claim 31 , wherein (b) comprises mechanically fragmenting said RNA molecule into said plurality of fragmented RNA molecules. 
     
     
         41 . The method of  claim 40 , wherein said mechanically fragmenting comprises use of sonication, nebulization, or pipetting. 
     
     
         42 . The method of  claim 31 , wherein (b) comprises using heat or light to fragment said RNA molecule into said plurality of fragmented RNA molecules. 
     
     
         43 . The method of  claim 31 , wherein said nucleic acid reaction comprises a ligation reaction. 
     
     
         44 . The method of  claim 31 , wherein said nucleic acid reaction comprises a nucleic acid extension reaction. 
     
     
         45 . The method of  claim 31 , further comprising performing a reverse transcription reaction on said fragmented RNA molecule to generate said derivative thereof. 
     
     
         46 . The method of  claim 31 , wherein said plurality of RNA molecules comprises messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), microRNA (miRNA), double stranded (dsRNA), a ribozyme, a riboswitch or a viral RNA. 
     
     
         47 . The method of  claim 31 , wherein (c) is performed in said partition. 
     
     
         48 . The method of  claim 31 , wherein said plurality of fragmented RNA molecules comprises non-overlapping fragmented RNA molecules. 
     
     
         49 . A method for processing a ribonucleic acid (RNA) molecule, comprising:
 (a) fragmenting an RNA molecule into a plurality of fragmented RNA molecules;   (b) partitioning fragmented RNA molecules of said plurality of fragmented RNA molecules, or derivatives thereof, to provide a partition comprising a bead comprising a nucleic acid barcode molecule coupled thereto; and   (c) subsequent to (b), using said nucleic acid barcode molecule and a fragmented RNA molecule of said fragmented RNA molecules, or derivative thereof, to perform a nucleic acid reaction to generate a barcoded RNA molecule.   
     
     
         50 . The method of  claim 49 , wherein said bead comprises a plurality of nucleic acid barcode molecules. 
     
     
         51 . The method of  claim 49 , wherein said partition is among a plurality of partitions. 
     
     
         52 . The method of  claim 51 , wherein said nucleic acid barcode molecule is different than other nucleic acid barcode molecules in other partitions of said plurality of partitions. 
     
     
         53 . The method of  claim 49 , wherein said partition is a droplet. 
     
     
         54 . The method of  claim 53 , wherein (b) comprises co-partitioning (i) said fragmented RNA molecules or derivatives thereof and (ii) said bead comprising said nucleic acid barcode molecule to provide said droplet. 
     
     
         55 . The method of  claim 49 , wherein said partition is a well. 
     
     
         56 . The method of  claim 49 , wherein said bead is a gel bead. 
     
     
         57 . The method of  claim 49 , wherein (a) comprises: (i) using a restriction enzyme to fragment said RNA molecule into said plurality of fragmented RNA molecules; (ii) mechanically fragmenting said RNA molecule into said plurality of fragmented RNA molecules; or (iii) using heat or light to fragment said RNA molecule into said plurality of fragmented RNA molecules. 
     
     
         58 . The method of  claim 49 , wherein said nucleic acid reaction comprises a ligation reaction. 
     
     
         59 . The method of  claim 49 , wherein said nucleic acid reaction comprises a nucleic acid extension reaction. 
     
     
         60 . The method of  claim 49 , wherein (c) is performed in said partition.