Compositions and methods for sample processing

Abstract

This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.

What is claimed is: 
     
         1 . A method for generating a plurality of barcode molecules, comprising:
 (a) providing a plurality of molecules coupled to a plurality of supports; and   (b) combinatorially assembling said plurality of barcode molecules coupled to said plurality of supports by coupling one or more molecules each comprising one or more segments to each of said plurality of molecules, wherein said plurality of barcode molecules comprises barcode molecules coupled to a support of said plurality of supports, and wherein said barcode molecules comprise barcode sequences that are different from other barcode sequences of other barcode molecules coupled to other supports of said plurality of supports.   
     
     
         2 . The method of  claim 1 , wherein said barcode sequences of said barcode molecules are identical. 
     
     
         3 . The method of  claim 1 , wherein said plurality of supports is a plurality of beads, and wherein said barcode molecules are coupled to a bead of said plurality of beads. 
     
     
         4 . The method of  claim 3 , wherein said at least said subset of said barcode molecules is coupled to an interior of said bead. 
     
     
         5 . The method of  claim 3 , wherein said barcode molecules are releasably coupled to said bead. 
     
     
         6 . The method of  claim 5 , wherein said barcode molecules are releasably coupled to said bead through chemical cross-linkers. 
     
     
         7 . The method of  claim 3 , wherein said plurality of beads is a plurality of gel beads. 
     
     
         8 . The method of  claim 3 , wherein said plurality of beads is dissolvable or disruptable 
     
     
         9 . The method of  claim 1 , wherein (b) comprises sequentially coupling multiple molecules to each of said plurality of molecules. 
     
     
         10 . The method of  claim 1 , wherein in (b) said one or more molecules comprise a first molecule comprising a first segment and a second molecule comprising a second segment, and wherein said combinatorially assembling comprises coupling said first molecule to a molecule of said plurality of molecules and coupling said second molecule to said first molecule, wherein said molecule is coupled to said support. 
     
     
         11 . The method of  claim 10 , further comprising coupling a third molecule comprising a third segment to said second molecule. 
     
     
         12 . The method of  claim 11 , wherein said third segment is different than said first segment or said second segment. 
     
     
         13 . The method of  claim 11 , wherein said third segment is different than said first segment and said second segment. 
     
     
         14 . The method of  claim 10 , wherein said first segment is different than said second segment. 
     
     
         15 . The method of  claim 1 , wherein (b) comprises ligating said one or more molecules to each of said plurality of molecules. 
     
     
         16 . The method of  claim 1 , wherein (b) comprises partitioning said plurality of molecules in separate partitions, and coupling said one or more molecules to each of said plurality of molecules in said separate partitions. 
     
     
         17 . The method of  claim 16 , wherein (b) further comprises partitioning said plurality of molecules in one or more additional partitions, and coupling at least a subset of said one or more molecules to said plurality of molecules in each of said one or more additional partitions. 
     
     
         18 . The method of  claim 17 , wherein said separate partitions are wells. 
     
     
         19 . The method of  claim 1 , wherein said barcode sequences are nucleic acid sequences. 
     
     
         20 . The method of  claim 1 , wherein said one or more segments are at least one nucleotide in length. 
     
     
         21 . The method of  claim 1 , wherein each of said barcode molecules comprises a unique identifier that is different than unique identifiers of other barcode molecules coupled to said support. 
     
     
         22 . The method of  claim 1 , wherein said barcode molecules comprise at least 100,000 barcode molecules. 
     
     
         23 . The method of  claim 1 , wherein each of said barcode molecules comprise a segment separated from said one or more segments by a sequence that is the same across at least a subset of said plurality of barcode molecules. 
     
     
         24 . The method of  claim 1 , wherein said one or more segments for at least a subset of said plurality of barcode molecules are different.