Analysis of nucleic acid sequences
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 of determining a presence of a structural variation of a nucleic acid, comprising:
(a) providing: (i) 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; and (ii) a plurality of oligonucleotides, wherein each oligonucleotide of the plurality of oligonucleotides comprises a barcode sequence and a random primer sequence, wherein the barcode sequence and the random primer sequence are on a same molecule;
(b) hybridizing random primer sequences of a subset of oligonucleotides of the plurality of oligonucleotides to multiple different locations of the given first fragment molecule, wherein each oligonucleotide of the subset of oligonucleotides comprises a common barcode sequence;
(c) generating a plurality of second fragment molecules comprising barcode sequences with the plurality of first fragment molecules and the plurality of oligonucleotides, wherein second fragment molecules of the plurality of second fragment molecules corresponding to the given first fragment molecule comprise the common barcode sequence and are generated by nucleic acid extension reactions using the random primer sequences of the subset of oligonucleotides and using the given first fragment molecule as a template;
(d) sequencing the plurality of second fragment molecules to provide a plurality of fragment sequences, wherein each of the plurality of fragment sequences corresponding to the given first fragment molecule shares the common barcode sequence; and
(e) determining a 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.
2. The method of claim 1 , wherein the elevated amount is 1% or more with respect to a total number of the first fragment molecules that are derived from a region of the nucleic acid having the structural variation.
3. The method of claim 1 , wherein the locations are at least 100 bases apart.
4. The method of claim 1 , further comprising identifying the structural variation by creating an assembly of the given first fragment molecule from the plurality of fragment sequences, wherein the plurality of fragment sequences are selected as inputs for the assembly based upon a presence of the common barcode sequence.
5. The method of claim 4 , further comprising creating the assembly by generating a consensus sequence from the plurality of fragment sequences.
6. The method of claim 1 , wherein the structural variation comprises a translocation.
7. A method of identifying variants in a sequence of a nucleic acid, comprising:
(a) providing: (i) a plurality of individual fragment molecules of the nucleic acid, and (ii) a plurality of oligonucleotides, wherein each oligonucleotide of the plurality of oligonucleotides comprises a random primer sequence;
(b) hybridizing random primer sequences of the plurality of oligonucleotides to multiple different locations of each individual fragment molecule of the plurality of individual fragment molecules;
(c) obtaining nucleic acid sequences of the plurality of individual fragment molecules of the nucleic acid by performing nucleic acid extension reactions using the random primer sequences of the plurality of oligonucleotides and the plurality of individual fragment molecules as templates, the nucleic acid sequences of the plurality of individual fragment molecules each having a length of at least 1 kilobase (kb);
(d) linking sequences of the nucleic acid sequences of the plurality of individual fragment molecules in one or more inferred contigs; and
(e) identifying one or more variants from the one or more inferred contigs.
8. The method of claim 7 , wherein (c) comprises:
generating a plurality of barcoded fragments of each individual fragment molecule of the plurality of individual fragment molecules, the barcoded fragments of a given individual fragment molecule having a common barcode;
sequencing the plurality of barcoded fragments of the plurality of individual fragment molecules, the sequencing providing a sequencing error rate of less than 1%; and
determining a sequence of the plurality of individual fragment molecules from sequences of the plurality of barcoded fragments and their associated barcodes.
9. The method of claim 8 , wherein the linking comprises identifying one or more overlapping sequences between two or more individual fragment molecules of the plurality of individual fragment molecules to link the two or more individual fragment molecules into the one or more inferred contigs.
10. The method of claim 9 , wherein the linking comprises identifying one or more common variants between two or more individual fragment molecules of the plurality of individual fragment molecules to link the two or more individual fragment molecules into the one or more inferred contigs.