Methods and compositions for targeted nucleic acid sequencing

Abstract

The present invention is directed to methods, compositions and systems for capturing and analyzing sequence information contained in targeted regions of a genome. Such targeted regions may include exomes, partial exomes, introns, combinations of exonic and intronic regions, genes, panels of genes, and any other subsets of a whole genome that may be of interest.

1 .- 97 . (canceled) 
     
     
         98 . A method for sequencing one or more selected portions of a genome, the method comprising:
 (a) distributing individual nucleic acid molecules from a starting genomic material into discrete partitions such that each discrete partition contains an individual nucleic acid molecule;   (b) fragmenting the individual nucleic acid molecules in the discrete partitions to form a plurality of fragments, wherein each of the fragments further comprises a barcode, wherein fragments within a given discrete partition each comprise a common barcode, and wherein each discrete partition is associated with a unique barcode, thereby associating each fragment with the individual nucleic acid molecule from which it is derived,   (c) enriching for a population of fragments comprising at least a portion of the one or more selected portions of the genome;   (d) obtaining sequence information comprising a plurality of sequence reads of the fragments from the population; and   (e) attributing a first sequence read of the plurality of sequence reads of the fragments to a same individual nucleic acid molecule in one of the discrete partitions as a second sequence read in the plurality of sequence reads, wherein the first and second sequence reads each map to genomic locations that are a distance from each other of longer than 1 kb.   
     
     
         99 . The method of  claim 98 , wherein at least one of the sequence reads of the plurality of sequence reads is linked to another sequence read in the plurality of sequence reads by a distance of longer than 10 kb on the genome. 
     
     
         100 . The method of  claim 98 , wherein (c) comprises:
 (i) hybridizing probes complementary to regions in or near the one or more selected portions of the genome to the fragments that comprise the selected portions of the genome to form probe-fragment complexes; and   (ii) capturing probe-fragment complexes to a surface of a solid support.   
     
     
         101 . The method of  claim 100 , wherein the probes comprise binding moieties and the surface comprises capture moieties, and wherein the probe-fragment complexes are captured on the surface through a reaction between the binding moieties and the capture moieties. 
     
     
         102 . The method of  claim 98 , wherein the enriching comprises one of the follow: whole or partial exome capture, panel capture, targeted exon capture, anchored exome capture, and tiled genomic region capture. 
     
     
         103 . The method of  claim 98 , wherein (d) comprises a sequencing reaction selected from the group consisting of: short read-length sequencing reactions and long read-length sequencing reactions. 
     
     
         104 . The method of  claim 98 , wherein (d) provides sequence information on less than 90% of the starting genomic material. 
     
     
         105 . The method of  claim 98 , wherein each discrete partition comprises genomic DNA from a different chromosome. 
     
     
         106 . The method of  claim 98 , wherein the barcode attached to the fragments is from a library of at least 700,000 barcodes. 
     
     
         107 . The method of  claim 98 , wherein subsequent to (b) and prior to (c), the method further comprises pooling the fragments from at least a subset of the discrete partitions. 
     
     
         108 . A method for sequencing one or more selected portions of a genome, the method comprising:
 (a) distributing individual nucleic acid molecules from a starting genomic material into discrete partitions such that each discrete partition contains an individual nucleic acid molecule;   (b) generating a plurality of barcoded fragments from the individual nucleic acid molecules in the discrete partitions, wherein fragments from the same individual nucleic acid molecule comprise a common barcode, and wherein each discrete partition is associated with a unique barcode, thereby associating each fragment with the individual nucleic acid molecule from which it is derived;   (c) enriching for a population of fragments comprising at least a portion of the one or more selected portions of the genome;   (d) sequencing fragments from the population;   (e) linking two or more of the individual nucleic acid molecules in a phase block based upon the sequences of overlapping phased variants within the sequences of the fragments from the population, wherein the phase block comprises a length N50 of at least 10 kb.   
     
     
         109 . The method of  claim 108 , wherein (c) comprises:
 (i) hybridizing probes complementary to regions in or near the one or more selected portions of the genome to the fragments that comprise the selected portions of the genome to form probe-fragment complexes; and   (ii) capturing probe-fragment complexes to a surface of a solid support.   
     
     
         110 . The method of  claim 109 , wherein the probes comprise binding moieties and the surface comprises capture moieties, and wherein the probe-fragment complexes are captured on the surface through a reaction between the binding moieties and the capture moieties. 
     
     
         111 . The method of  claim 108 , wherein the enriching comprises one of the follow: whole or partial exome capture, panel capture, targeted exon capture, anchored exome capture, and tiled genomic region capture. 
     
     
         112 . The method of  claim 108 , wherein (d) comprises a sequencing reaction selected from the group consisting of: short read-length sequencing reactions and long read-length sequencing reactions. 
     
     
         113 . The method of  claim 108 , wherein (d) provides sequence information on less than 90% of the starting genomic material. 
     
     
         114 . The method of  claim 108 , wherein each discrete partition comprises genomic DNA from a different chromosome. 
     
     
         115 . The method of  claim 108 , wherein the barcode attached to the fragments is from a library of at least 700,000 barcodes. 
     
     
         116 . The method of  claim 108 , wherein subsequent to (b) and prior to (c), the method further comprises pooling the fragments from the discrete partitions. 
     
     
         117 . A method of obtaining sequence information from one or more targeted portions of a genomic sample, the method comprising:
 (a) providing individual nucleic acid molecules from the genomic sample in discrete partitions, wherein the individual nucleic acid molecules have lengths greater than 1 kilobase (kb);   (b) providing a plurality of barcode sequences in the discrete partitions, wherein the plurality of barcode sequences comprises at least 100,000 different sequences;   (c) generating a plurality of barcoded fragments from the individual nucleic acid molecules in the discrete partitions, wherein fragments from the same individual nucleic acid molecule comprise a common barcode, and wherein each discrete partition is associated with a unique barcode, thereby associating each fragment with the individual nucleic acid molecule from which it is derived;   (d) enriching the plurality of barcoded fragments for barcoded fragments comprising sequences associated with the one or more targeted portions of the genomic sample, thereby producing an enriched plurality of fragments; and   (e) conducting a sequencing reaction to identify sequences of the enriched plurality of fragments, thereby obtaining sequence information from the one or more targeted portions of the genomic sample.   
     
     
         118 . The method of  claim 117 , wherein the enriching in (d) is performed by:
 (i) hybridizing probes complementary to regions in or near the one or more selected portions of the genome to the fragments that comprise the selected portions of the genome to form probe-fragment complexes; and   (ii) capturing probe-fragment complexes to a surface of a solid support.   
     
     
         119 . The method of  claim 118 , wherein the probes comprise binding moieties and the surface comprises capture moieties, and wherein the probe-fragment complexes are captured on the surface through a reaction between the binding moieties and the capture moieties. 
     
     
         120 . The method of  claim 117 , wherein the enriching comprises one of the follow: whole or partial exome capture, panel capture, targeted exon capture, anchored exome capture, and tiled genomic region capture. 
     
     
         121 . The method of  claim 117 , wherein (e) comprises a sequencing reaction selected from the group consisting of: short read-length sequencing reactions and long read-length sequencing reactions. 
     
     
         122 . The method of  claim 117 , wherein (e) provides sequence information on less than 90% of the genomic sample. 
     
     
         123 . The method of  claim 117 , wherein the barcode attached to the fragments is from a library of at least 700,000 barcodes. 
     
     
         124 . The method of  claim 117 , wherein the individual nucleic acid molecules have lengths greater than 10 kb. 
     
     
         125 . The method of  claim 117 , wherein the method further comprises (f) determining linkage of two or more targeted portions of the genomic sample based upon common barcode sequences. 
     
     
         126 . The method of  claim 117 , wherein subsequent to (c) and prior to (d), the method further comprises pooling the barcoded fragments from the discrete partitions.