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 . A method for sequencing one or more selected portions of a genome, the method comprising:
 (a) providing starting genomic material;   (b) distributing individual nucleic acid molecules from the starting genomic material into discrete partitions such that each discrete partition contains a first individual nucleic acid molecule;   (c) 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, and wherein fragments within a given discrete partition each comprise a common barcode, thereby associating each fragment with the individual nucleic acid molecule from which it is derived;   (d) providing a population enriched for fragments comprising at least a portion of the one or more selected portions of the genome;   (e) obtaining sequence information from the population, thereby sequencing one or more selected portions of a genome.   
     
     
         2 . The method of  claim 1 , wherein the providing step (d) comprises:
 (i) hybridizing probes complementary to regions in or near the one or more selected portions of the genome to the fragments to form probe-fragment complexes;   (ii) capturing probe-fragment complexes to a surface of a solid support;   thereby enriching the population with fragments comprising at least a portion of the one or more selected portions of the genome.   
     
     
         3 . The method of  claim 2 , wherein the solid support comprises a bead. 
     
     
         4 . The method of  claim 2 , 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. 
     
     
         5 . The method of  claim 4 , wherein the capture moieties comprise streptavidin and the binding moieties comprise biotin. 
     
     
         6 . The method of  claim 4 , wherein the capture moieties comprise streptavidin magnetic beads and the binding moieties comprise biotinylated RNA library baits. 
     
     
         7 . The method of  claim 4 , wherein the capture moieties are directed to a member selected from the group consisting of: whole or partial exome capture, panel capture, targeted exon capture, anchored exome capture, and tiled genomic region capture. 
     
     
         8 . The method of  claim 1 , wherein prior to the obtaining step (e), the fragments are amplified to form amplification products. 
     
     
         9 . The method of  claim 8 , wherein the amplification products are capable of forming partial or complete hairpin structures. 
     
     
         10 . The method of  claim 1 , wherein the obtaining step (e) comprises a sequencing reaction selected from the group consisting of: short read-length sequencing reactions and long read-length sequencing reactions. 
     
     
         11 . The method of  claim 10 , wherein the sequencing reaction is a short read, high accuracy sequencing reaction. 
     
     
         12 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the method further comprises linking two or more of the individual nucleic acid molecules in an inferred contig based upon overlapping sequences of the isolated fragments, wherein the inferred contig comprises a length N50 of at least 10 kb. 
     
     
         16 . The method of  claim 15 , wherein the inferred contig comprises a length N50 of at least 20 kb. 
     
     
         17 . The method of  claim 15 , wherein the inferred contig comprises a length N50 of at least 40 kb. 
     
     
         18 . The method of  claim 15 , wherein the inferred contig comprises a length N50 of at least 50 kb. 
     
     
         19 . The method of  claim 15 , wherein the inferred contig comprises a length N50 of at least 100 kb. 
     
     
         20 . The method of  claim 15 , wherein the inferred contig comprises a length N50 of at least 200 kb. 
     
     
         21 . The method of  claim 1 , wherein the method further comprises linking two or more of the individual nucleic acid molecules in a phase block based upon overlapping phased variants within the sequences of the isolated fragments, wherein the phase block comprises a length N50 of at least 10 kb. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the selected portions of the genome comprise an exome. 
     
     
         28 . The method of  claim 1 , wherein the individual nucleic acid molecules in each discrete partition comprises genomic DNA from a single cell. 
     
     
         29 . The method of  claim 1 , wherein each discrete partition comprises genomic DNA from a different chromosome. 
     
     
         30 . The method of  claim 1 , wherein the discrete partitions comprise droplets in an emulsion. 
     
     
         31 . The method of  claim 1 , wherein the barcode attached to the fragments is from a library of at least 700,000 barcodes. 
     
     
         32 . The method of  claim 1 , wherein the barcode further comprises additional sequence segments. 
     
     
         33 . The method of  claim 32 , wherein the additional sequence segments comprise one or more of a member selected from the group consisting of: primers, attachment sequences, random n-mer oligonucleotides, oligonucleotides comprising uracil nucleobases. 
     
     
         34 . A method of obtaining sequence information from one or more targeted portions of a genomic sample, the method comprising:
 (a) providing individual first nucleic acid fragment molecules of the genomic sample in discrete partitions;   (b) fragmenting the individual first nucleic acid fragment molecules within the discrete partitions to create a plurality of second fragments from each of the individual first nucleic acid fragment molecules;   (c) attaching a common barcode sequence to the plurality of the second fragments within a discrete partition, such that each of the plurality of second fragments are attributable to the discrete partition in which they are contained;   (d) applying a library of probes directed to the one or more targeted portions of the genomic sample to the second fragments;   (e) conducting a sequencing reaction to identify sequences of the plurality of second fragments that hybridized to the library of probes, thereby obtaining sequence information from the one or more targeted portions of the genomic sample.   
     
     
         35 - 59 . (canceled) 
     
     
         60 . A method for obtaining sequence information from one or more targeted portions of a genomic sample while retaining molecular context, the method comprising
 (a) providing starting genomic material;   (b) distributing individual nucleic acid molecules from the starting genomic material into discrete partitions such that each discrete partition contains a first individual nucleic acid molecule;   (c) fragmenting the first individual nucleic acid molecules in the discrete partitions to form a plurality of fragments;   (d) providing a population enriched for fragments comprising at least a portion of the one or more selected portions of the genome;   (e) obtaining sequence information from the population, thereby sequencing one or more targeted portions of the genomic sample while retaining molecular context.   
     
     
         61 - 62 . (canceled) 
     
     
         63 . 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 of the genomic sample in discrete partitions;   (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, and wherein fragments within a given discrete partition each comprise a common barcode, thereby associating each fragment with the individual nucleic acid molecule from which it is derived;   (c) applying a library of probes directed to the one or more targeted portions of the genomic sample to the plurality of fragments, wherein at least a majority of the probes in the library of probes are designed to hybridize to informative single nucleotide polymorphisms (SNPs);   (d) conducting a sequencing reaction to identify sequences of the plurality of fragments that hybridized to the library of probes, thereby obtaining sequence information from the one or more targeted portions of the genomic sample.   
     
     
         64 . The method of  claim 63 , wherein about 80%-99% of the probes in the library of probes are designed to hybridize to informative SNPs. 
     
     
         65 - 85 . (canceled) 
     
     
         86 . 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 of the genomic sample;   (b) fragmenting the individual nucleic acid molecules to form a plurality of fragments, wherein each of the fragments further comprises a barcode, and wherein fragments from the same individual nucleic molecule comprise a common barcode, thereby associating each fragment with the individual nucleic acid molecule from which it is derived;   (c) enriching the plurality of fragments for fragments containing the one or more targeted portions of the genomic sample;   (d) 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.   
     
     
         87 . The method of  claim 86 , wherein barcodes are added to the individual nucleic acid molecules prior to the fragmenting step (b). 
     
     
         88 . The method of  claim 87 , wherein the barcodes are added to the individual nucleic acid molecules using a transposon. 
     
     
         89 . The method of  claim 86 , wherein barcodes are added simultaneously with the fragmenting. 
     
     
         90 . The method of  claim 89 , wherein the fragmenting comprises an amplification step. 
     
     
         91 . The method of  claim 86 , wherein the enriching step (c) comprises applying a library of probes directed to the one or more targeted portions of the genomic sample. 
     
     
         92 . The method of  claim 91 , wherein the library of probes are attached to binding moieties, and wherein prior to the conducting step (d), the fragments are captured through a reaction between the binding moieties and the capture moieties. 
     
     
         93 . The method of  claim 92 , wherein the reaction between the binding moieties and the capture moieties immobilizes the fragments on a surface. 
     
     
         94 . The method of  claim 92 , wherein the binding moieties comprise biotin and the capture moieties comprise streptavidin. 
     
     
         95 . The method of  claim 86 , wherein the sequencing reaction is a member selected from the group consisting of: short read-length sequencing reactions and long read-length sequencing reactions. 
     
     
         96 . The method of  claim 86 , wherein the sequencing reaction is a short read, high accuracy sequencing reaction. 
     
     
         97 . The method of  claim 86 , wherein the fragments are amplified such that the resultant amplification products are capable of forming partial or complete hairpin structures.