Methods for assembling and reading nucleic acid sequences from mixed populations

Abstract

The disclosure relates to methods for obtaining nucleic acid sequence information by constructing a nucleic acid library and reconstructing longer nucleic acid sequences by assembling a series of shorter nucleic acid sequences.

1 .- 82 . (canceled) 
     
     
         83 . A method for processing nucleic acid molecules from a biological sample for synthetic long-read sequencing, comprising:
 (a) providing a reaction chamber comprising said nucleic acid molecules derived from said biological sample, wherein a given nucleic acid molecule of said nucleic acid molecules has a length of at least about 500 bases;   (b) in said reaction chamber, bringing said nucleic acid molecules in contact with nucleic acid barcodes comprising different barcode sequences, under conditions sufficient for attachment of said nucleic acid barcodes to said nucleic acid molecules, thereby generating barcoded nucleic acid molecules, wherein a given barcoded nucleic acid molecule of said barcoded nucleic acid molecules has a different barcode sequence than other barcoded nucleic acid molecules of said barcoded nucleic acid molecules; and   (c) in said reaction chamber, subjecting said barcoded nucleic acid molecules to nucleic acid amplification, thereby generating amplification products of said barcoded nucleic acid molecules.   
     
     
         84 . The method of  claim 83 , wherein a given nucleic acid barcode of said nucleic acid barcodes is part of an oligonucleotide adapter. 
     
     
         85 . The method of  claim 84 , wherein said oligonucleotide adapter comprises a polymerase chain reaction (PCR) primer region. 
     
     
         86 . The method of  claim 85 , wherein (c) comprises hybridizing a primer to said PCR primer region, and wherein said nucleic acid amplification comprises PCR. 
     
     
         87 . The method of  claim 84 , wherein said oligonucleotide adapter further comprises a sequencing primer region. 
     
     
         88 . The method of  claim 83 , wherein said nucleic acid barcodes comprise an identical sample identifier sequence that identifies said barcoded nucleic acid molecules with said biological sample. 
     
     
         89 . The method of  claim 83 , wherein attachment of said nucleic acid barcodes to said nucleic acid molecules is performed by ligation. 
     
     
         90 . The method of  claim 83 , further comprising, subsequent to (c), fragmenting said amplification products of said barcoded nucleic acid molecules in said reaction chamber, thereby generating barcoded nucleic acid fragments comprising a given nucleic acid barcode at a first end and a portion of a given one of said nucleic acid molecules at a second end. 
     
     
         91 . The method of  claim 90 , wherein said fragmenting comprises random fragmenting. 
     
     
         92 . The method of  claim 90 , wherein said fragmenting is performed by an enzyme. 
     
     
         93 . The method of  claim 90 , further comprising circularizing said barcoded nucleic acid fragments in said reaction chamber, thereby generating circularized nucleic acids comprising said given nucleic acid barcode adjacent to said portion of said given one of said nucleic acid molecules. 
     
     
         94 . The method of  claim 93 , further comprising fragmenting said circularized nucleic acids in said reaction chamber, thereby generating linear barcoded fragments. 
     
     
         95 . The method of  claim 94 , further comprising attaching an oligonucleotide adapter to said linear barcoded fragments, thereby generating linear barcoded fragments comprising said oligonucleotide adapter. 
     
     
         96 . The method of  claim 83 , further comprising sequencing said barcoded nucleic acid molecules or derivatives thereof, to yield nucleic acid sequence information for said barcoded nucleic acid molecules or derivatives thereof 
     
     
         97 . The method of  claim 96 , wherein said nucleic acid sequence information comprises greater than about 95% fidelity to a target sequence in said nucleic acid molecules. 
     
     
         98 . The method of  claim 96 , further comprising assembling said nucleic acid sequence information for said barcoded nucleic acid molecules or derivatives thereof to generate a contiguous sequence of a given nucleic acid molecule of said nucleic acid molecules derived from said biological sample. 
     
     
         99 . The method of  claim 83 , further comprising, prior to (a), fragmenting a plurality of nucleic acids derived from said biological sample, thereby generating said nucleic acid molecules. 
     
     
         100 . The method of  claim 83 , wherein (a) comprises providing a plurality of reaction chambers each comprising nucleic acid molecules derived from a plurality of different biological samples including said biological sample, and wherein (b) and (c) are performed in each of said plurality of reaction chambers to generate amplification products or derivatives thereof in each of said plurality of reaction chambers. 
     
     
         101 . The method of  claim 100 , wherein said amplification products or derivatives thereof comprise sample identifier sequences that identify said amplification products as having been derived from a given biological sample of said plurality of different biological samples. 
     
     
         102 . The method of  claim 101 , further comprising pooling said amplification products or derivatives thereof from said plurality of reaction chambers, and subjecting said amplification products or derivatives thereof to sequencing.