Analysis of nucleic acid sequences

Abstract

The present disclosure relates to methods, compositions and systems for haplotype phasing and copy number variation assays. Included within this disclosure are methods and systems for combining the barcode comprising beads with samples in multiple separate partitions, as well as methods of processing, sequencing and analyzing barcoded samples.

1 . A method for identifying one or more variations in a nucleic acid, comprising:
 (a) providing a first fragment of the nucleic acid, wherein the first fragment has a length greater than 10 kilobases (kb);   (b) sequencing a plurality of second fragments of the first fragment to provide a plurality of fragment sequences, which plurality of fragment sequences share a common barcode sequence;   (c) attributing the plurality of fragment sequences to the first fragment by a presence of the common barcode sequence;   (d) determining a nucleic acid sequence of the first fragment using the plurality of fragment sequences, wherein the nucleic acid sequence is determined at an error rate of less than 1%; and   (e) identifying the one or more variations in the nucleic acid sequence of the first fragment determined in (d), thereby identifying the one or more variations within the nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein the first fragment is in a discrete partition among a plurality of discrete partitions. 
     
     
         3 . The method of  claim 2 , wherein the discrete partition is a droplet in an emulsion. 
     
     
         4 . The method of  claim 1 , wherein the identifying comprises identifying phased variants in the nucleic acid from the nucleic acid sequence of the first fragment. 
     
     
         5 . The method of  claim 1 , wherein the identifying comprises identifying one or more structural variations in the nucleic acid from the nucleic acid sequence of the first fragment. 
     
     
         6 . The method of  claim 1 , wherein the first fragment has a length greater than 15 kb. 
     
     
         7 . The method of  claim 1 , wherein the first fragment has a length greater than 20 kb. 
     
     
         8 . The method of  claim 1 , wherein the determining comprises mapping the plurality of fragment sequences to a reference. 
     
     
         9 . The method of  claim 1 , wherein the determining comprises assembling the plurality of fragment sequences with the common barcode sequence. 
     
     
         10 . The method of  claim 1 , further comprising providing a plurality of first fragments of the nucleic acid that are at least 10 kb in length, and wherein the identifying comprises determining a nucleic acid sequence from each of the plurality of first fragments and identifying the one or more variations in the nucleic acid from the nucleic acid sequence from each of the plurality of first fragments. 
     
     
         11 . The method of  claim 10 , further comprising linking two or more nucleic acid sequences of the plurality of first fragments in an inferred contig based upon overlapping nucleic acid sequences of the two or more nucleic acid sequences, wherein the maximum inferred contig length is at least 10 kb. 
     
     
         12 . The method of  claim 11 , wherein the maximum inferred contig length is at least 20 kb. 
     
     
         13 . The method of  claim 12 , wherein the maximum inferred contig length is at least 40 kb. 
     
     
         14 . The method of  claim 13  wherein the maximum inferred contig length is at least 50 kb. 
     
     
         15 . The method of  claim 14 , wherein the maximum inferred contig length is at least 100 kb. 
     
     
         16 . The method of  claim 15 , wherein the maximum inferred contig length is at least 200 kb. 
     
     
         17 . The method of  claim 16 , wherein the maximum inferred contig length is at least 500 kb. 
     
     
         18 . The method of  claim 17 , wherein the maximum inferred contig length is at least 750 kb. 
     
     
         19 . The method of  claim 18 , wherein the maximum inferred contig length is at least 1 megabase (Mb). 
     
     
         20 . The method of  claim 19 , wherein the maximum inferred contig length is at least 1.75 Mb. 
     
     
         21 . The method of  claim 20 , wherein the maximum inferred contig length is at least 2.5 Mb. 
     
     
         22 . The method of  claim 10 , further comprising linking two or more nucleic acid sequences of the plurality of first fragments in a phase block based upon overlapping phased variants within the two or more nucleic acid sequences of the plurality of first fragments, wherein the maximum phase block length is at least 10 kb. 
     
     
         23 . The method of  claim 22 , wherein the maximum phase block length is at least 20 kb. 
     
     
         24 . The method of  claim 23 , wherein the maximum phase block length is at least 40 kb. 
     
     
         25 . The method of  claim 24 , wherein the maximum phase block length is at least 50 kb. 
     
     
         26 . The method of  claim 25 , wherein the maximum phase block length is at least 100 kb. 
     
     
         27 . The method of  claim 26 , wherein the maximum phase block length is at least 200 kb. 
     
     
         28 . The method of  claim 27 , wherein the maximum phase block length is at least 500 kb. 
     
     
         29 . The method of  claim 28 , wherein the maximum phase block length is at least 750 kb. 
     
     
         30 . The method of  claim 29 , wherein the maximum phase block length is at least 1 Mb. 
     
     
         31 . The method of  claim 30 , wherein the maximum phase block length is at least 1.75 Mb. 
     
     
         32 . The method of  claim 31 , wherein the maximum phase block length is at least 2.5 Mb. 
     
     
         33 . The method of  claim 10 , further comprising linking two or more nucleic acid sequences of the plurality of first fragments in an inferred contig based upon overlapping nucleic acid sequences of the two or more nucleic acid sequences, thereby creating a population of inferred contigs, wherein the N50 of the population of inferred contigs is at least 10 kb. 
     
     
         34 . The method of  claim 33 , wherein the N50 of the population of inferred contigs is at least 20 kb. 
     
     
         35 . The method of  claim 34 , wherein the N50 of the population of inferred contigs is at least 40 kb. 
     
     
         36 . The method of  claim 35 , wherein the N50 of the population of inferred contigs is at least 50 kb. 
     
     
         37 . The method of  claim 36 , wherein the N50 of the population of inferred contigs is at least 100 kb. 
     
     
         38 . The method of  claim 37 , wherein the N50 of the population of inferred contigs is at least 200 kb. 
     
     
         39 . The method of  claim 38 , wherein the N50 of the population of inferred contigs is at least 500 kb. 
     
     
         40 . The method of  claim 39 , wherein the N50 of the population of inferred contigs is at least 750 kb. 
     
     
         41 . The method of  claim 40 , wherein the N50 of the population of inferred contigs is at least 1 Mb. 
     
     
         42 . The method of  claim 41 , wherein the N50 of the population of inferred contigs is at least 1.75 Mb. 
     
     
         43 . The method of  claim 42 , wherein the N50 of the population of inferred contigs is at least 2.5 Mb. 
     
     
         44 . The method of  claim 10 , further comprising linking two or more nucleic acid sequences of the plurality of first fragments in a phase block based upon overlapping phased variants within the two or more nucleic acid sequences of the plurality of first fragments, thereby creating a population of phase blocks, wherein the N50 of the population of phase blocks is at least 10 kb. 
     
     
         45 . The method of  claim 44 , wherein the N50 of the population of phase blocks is at least 20 kb. 
     
     
         46 . The method of  claim 45 , wherein the N50 of the population of phase blocks is at least 40 kb. 
     
     
         47 . The method of  claim 46 , wherein the N50 of the population of phase blocks is at least 50 kb. 
     
     
         48 . The method of  claim 47 , wherein the N50 of the population of phase blocks is at least 100 kb. 
     
     
         49 . The method of  claim 48 , wherein the N50 of the population of phase blocks is at least 200 kb. 
     
     
         50 . The method of  claim 49 , wherein the N50 of the population of phase blocks is at least 500 kb. 
     
     
         51 . The method of  claim 50 , wherein the N50 of the population of phase blocks is at least 750 kb. 
     
     
         52 . The method of  claim 51 , wherein the N50 of the population of phase blocks is at least 1 Mb. 
     
     
         53 . The method of  claim 52 , wherein the N50 of the population of phase blocks is at least 1.75 Mb. 
     
     
         54 . The method of  claim 53 , wherein the N50 of the population of phase blocks is at least 2.5 Mb. 
     
     
         55 . A method of determining a presence of a structural variation of a nucleic acid, comprising:
 (a) providing a plurality of first fragment molecules of the nucleic acid, wherein a given first fragment molecule of the plurality of first fragment molecules comprises the structural variation;   (b) sequencing a plurality of second fragment molecules of each of the plurality of first fragment molecules to provide a plurality of fragment sequences, wherein each of the plurality of fragment sequences corresponding to a given first fragment molecule shares a common barcode sequence; and   (c) determining the presence of the structural variation by (i) mapping the plurality of fragment sequences to a reference sequence, (ii) identifying the plurality of fragment sequences that share the common barcode sequence, and (iii) identifying the structural variation based on a presence of an elevated amount of the plurality of fragment sequences sharing the common barcode sequence that map to the reference sequence at locations that are further apart than a length of the given first fragment molecule, which elevated amount is relative to a sequence lacking the structural variation.   
     
     
         56 .- 64 . (canceled) 
     
     
         65 . A method of characterizing a variant nucleic acid sequence, comprising:
 (a) fragmenting a variant nucleic acid to provide a plurality of first fragments having a length greater than 10 kilobases (kb);   (b) separating the plurality of first fragments into discrete partitions;   (c) creating a plurality of second fragments from each first fragment within its respective partition, the plurality of second fragments having a barcode sequence attached thereto, which barcode sequence within a given partition is a common barcode sequence;   (d) sequencing the plurality of second fragments and the barcode sequences attached thereto, to provide a plurality of second fragment sequences;   (e) attributing the second fragment sequences to an original first fragment based at least in part on the presence of the common barcode sequence to provide a first fragment sequence context for the second fragment sequences; and   (f) identifying a variant portion of the variant nucleic acid from the first fragment sequence context, thereby characterizing the variant nucleic acid sequence.   
     
     
         66 .- 73 . (canceled) 
     
     
         74 . A method of identifying variants in a sequence of a nucleic acid, comprising:
 obtaining nucleic acid sequences of a plurality of individual fragment molecules of the nucleic acid, the nucleic acid sequences of the plurality of individual fragment molecules each having a length of at least 1 kilobase (kb);   linking sequences of one or more of the plurality of individual fragment molecules in one or more inferred contigs; and   identifying one or more variants from the one or more inferred contigs.   
     
     
         75 .- 81 . (canceled) 
     
     
         82 . A method of characterizing nucleic acids, comprising:
 obtaining nucleic acid sequences of a plurality of fragment molecules having a length of at least 10 kilobases (kb);   identifying one or more phased variant positions in the nucleic acid sequences of the plurality of fragment molecules;   linking the nucleic acid sequences of at least a first fragment molecule to at least a second fragment molecule based upon a presence of one or more common phased variant positions within the first and second fragment molecules, to provide a phase block with a maximum phase block length of at least 10 kb; and   identifying one or more phased variants from the phase block with the maximum phase block length of at least 10 kb.   
     
     
         83 .- 92 . (canceled) 
     
     
         93 . A method, comprising:
 a) partitioning a first nucleic acid into a first partition, where the first nucleic acid comprises the target sequence derived from a first chromosome of an organism;   b) partitioning a second nucleic acid into a second partition, where the second nucleic acid comprises the target sequence derived from a second chromosome of the organism;   c) in the first partition, attaching a first barcode sequence to fragments of the first nucleic acid or to copies of portions of the first nucleic acid to provide first barcoded fragments;   d) in the second partition, attaching a second barcode sequence to fragments of the second nucleic acid or to copies of portions of the second nucleic acid to provide second barcoded fragments, the second barcode sequence being different from the first barcode sequence;   e) determining the nucleic acid sequence of the first and second barcoded fragments, and assembling a nucleic acid sequence of the first and second nucleic acids; and   f) comparing the nucleic acid sequence of the first and second nucleic acids to characterize the first and second nucleic acids as deriving from first and second chromosomes, respectively.   
     
     
         94 .- 99 . (canceled) 
     
     
         100 . A method, comprising:
 a) partitioning a first nucleic acid into a first partition, where the first nucleic acid comprises the target sequence derived from a first chromosome of an organism;   b) partitioning a second nucleic acid into a second partition, where the second nucleic acid comprises the target sequence derived from a second chromosome of the organism;   c) in the first partition, attaching a first barcode sequence to fragments of the first nucleic acid or to copies of portions of the first nucleic acid to provide first barcoded fragments;   d) in the second partition, attaching a second barcode sequence to fragments of the second nucleic acid or to copies of portions of the second nucleic acid to provide second barcoded fragments, the second barcode sequence being different from the first barcode sequence;   e) determining the nucleic acid sequence of the first and second barcoded fragments, and assembling a nucleic acid sequence of the first and second nucleic acids; and   f) comparing the nucleic acid sequence of the first and second nucleic acids to identify any variation between the nucleic acid sequence of the first and second nucleic acids.   
     
     
         101 .- 116 . (canceled) 
     
     
         117 . A method for characterizing a fetal nucleic acid sequence, comprising:
 (a) determining a maternal nucleic acid sequence, wherein the maternal nucleic acid is derived from a pregnant mother of a fetus, by:
 (i) fragmenting a maternal nucleic acid to provide a plurality of first maternal fragments; 
 (ii) separating the plurality of first maternal fragments into maternal partitions; 
 (iii) creating a plurality of second maternal fragments from each of the first maternal fragments within their respective maternal partitions, the plurality of second maternal fragments having a first barcode sequence attached thereto, wherein within a given maternal partition of the maternal partitions the second maternal fragments comprise a first common barcode sequence attached thereto; 
 (iv) sequencing the plurality of second maternal fragments to provide a plurality of maternal fragment sequences; and 
 (v) attributing the maternal fragment sequences to an original first maternal fragment based at least in part on the presence of the first common barcode sequence to determine the maternal nucleic acid sequence; 
   (b) determining a paternal nucleic acid sequence, wherein the paternal nucleic acid is derived from a father of the fetus, by:
 (i) fragmenting a paternal nucleic acid to provide a plurality of first paternal fragments; 
 (ii) separating the plurality of first paternal fragments into paternal discrete partitions; 
 (iii) creating a plurality of second paternal fragments from each first paternal fragment within its respective partition, the plurality of second paternal fragments having a second barcode sequence attached thereto, wherein within a given paternal partition, the second paternal fragments comprise a second common barcode sequence attached thereto; 
 (iv) sequencing the plurality of second paternal fragments and the second barcode sequences attached thereto, to provide a plurality of paternal fragment sequences; and 
 (v) attributing the paternal fragment sequences to an original first paternal fragment based at least in part on the presence of the second common barcode sequence to determine the paternal nucleic acid sequence; and 
   (c) obtaining a fetal nucleic acid from the pregnant mother and determining a sequence of the fetal nucleic acid and/or one or more genetic variations of the sequence of the fetal nucleic acid using the maternal nucleic acid sequence and the paternal nucleic acid sequence.   
     
     
         118 .- 139 . (canceled) 
     
     
         140 . A method for characterizing a sample nucleic acid, comprising:
 (a) obtaining a biological sample from a subject, which biological sample includes a cell-free sample nucleic acid;   (b) in a droplet, attaching a barcode sequence to fragments of the cell-free sample nucleic acid or to copies of portions of the sample nucleic acid, to provide barcoded sample fragments;   (c) determining nucleic acid sequences of the barcoded sample fragments and providing a sample nucleic acid sequence based on the nucleic acid sequences of the barcoded sample fragments;   (d) using a programmed computer processor to generate a comparison of the sample nucleic acid sequence to a reference nucleic acid sequence, which reference nucleic acid sequence has a length greater 10 kilobases (kb) and an accuracy of at least 99%; and   (e) using the comparison to identify one or more genetic variations in the sample nucleic acid sequence, thereby associating the sample nucleic acid with a disease.   
     
     
         141 .- 163 . (canceled)