Methods and Compositions for Targeted Nucleic Acid Sequence Coverage

Abstract

The present invention is directed to methods, compositions and systems for analyzing sequence information from targeted regions of a genome. Such targeted regions may include regions of the genome that are poorly characterized, highly polymorphic, or divergent from reference genome sequences.

What is claimed: 
     
         1 .- 93 . (canceled) 
     
     
         94 . A method for obtaining sequence information from one or more portions of a genomic sample while retaining molecular context, the method comprising
 (a) providing starting genomic material;   (b) distributing a plurality of individual nucleic acid molecules from the starting genomic material into discrete partitions, wherein the genomic material within each discrete partition comprises genomic DNA from a single cell;   (c) amplifying one or more targeted portions of at least some of the individual nucleic acid molecules in the discrete partitions to form a plurality of amplicons, wherein the amplifying is conducted with a library of primer pairs and wherein the library of primer pairs is designed to amplify at specific distances along the targeted portions;   (d) simultaneously with or following step (c), attaching a common barcode sequence to the amplicons within each discrete partition such that each of the amplicons is attributable to the discrete partition in which it was contained;   (e) obtaining sequence information from the amplicons, thereby sequencing one or more targeted portions of the genomic sample while retaining molecular context, wherein the library of primer pairs is selected to amplify genomic regions that are poorly characterized, highly polymorphic, or divergent from reference genome sequences.   
     
     
         95 . The method of  claim 94 , wherein the one or more targeted portions comprise a contiguous region of the genome of at least 3.0 Mb in length. 
     
     
         96 . The method of claim  1  or claim  2 , 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. 
     
     
         97 . The method of any one of claims  1  to  3 , wherein the identifying step (e) preserves the molecular context of the sequences of the amplicons, such that the identifying further comprises identifying amplicons derived from the same first individual nucleic acid molecules. 
     
     
         98 . The method of any one of claims  1  to  4 , wherein the method further comprises linking two or more of the individual first fragment molecules in an inferred contig based upon overlapping sequences of the plurality of amplicons, wherein the inferred contig comprises a length N50 of at least 10 kb. 
     
     
         99 . The method of  claim 98 , wherein the inferred contig comprises a length N50 of at least 50 kb. 
     
     
         100 . The method of  claim 98 , wherein the inferred contig comprises a length N50 of at least 100 kb. 
     
     
         101 . The method of  claim 98 , wherein the inferred contig comprises a length N50 of at least 200 kb. 
     
     
         102 . The method of any one of claims  1  to  5 , wherein the barcode sequence further comprises additional sequence segments. 
     
     
         103 . The method of claim  6 , 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. 
     
     
         104 . The method of any one of claims  1  to  7 , wherein the attaching step (d) comprises attaching a barcode selected from a library of at least 700,000 barcodes. 
     
     
         105 . The method of any one of claims  1  to  8 , wherein the discrete partitions comprise droplets in an emulsion. 
     
     
         106 . The method of any one of claims  1  to  9 , wherein prior to the obtaining step (e), the amplicons are further amplified such that the resultant amplification products are capable of forming partial or complete hairpin structures. 
     
     
         107 . The method of any one of claims  1  to  10 , wherein the barcoded amplicons within the discrete partitions represent about 100×-5000× coverage of the one or more targeted portions. 
     
     
         108 . The method of any one of claims  1  to  10 , wherein the barcoded amplicons within the discrete partitions represent about 200×-1000× coverage of the one or more targeted portions. 
     
     
         109 . The method of any one of claims  1  to  10 , wherein the barcoded amplicons within the discrete partitions represent at least 1× coverage of the one or more targeted portions. 
     
     
         110 . The method of any one of claims  1  to  10 , wherein the barcoded amplicons within the discrete partitions represent at least 2000× coverage of the one or more targeted portions. 
     
     
         111 . The method of any one of claims  1  to  10 , wherein the barcoded amplicons within the discrete partitions represent at least 5000× coverage of the one or more targeted portions. 
     
     
         112 . The method of  claim 94 , wherein the library of primer pairs is designed to amplify across a region of at least 3.5 megabases (Mb). 
     
     
         113 . The method of  claim 94 , wherein the library of primer is designed to stagger across a region of at least 3.0 megabases (Mb).