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 for nucleic acid analysis, comprising:
(a) generating a plurality of barcoded parental nucleic acid molecules in a plurality of partitions using (i) parental nucleic acid molecules derived from a parental biological sample and (ii) nucleic acid barcode molecules from a plurality of nucleic acid barcode molecules;
(b) removing or releasing said plurality of barcoded parental nucleic acid molecules from said plurality of partitions;
(c) using said barcoded parental nucleic acid molecules or derivatives thereof to generate parental nucleic acid sequence information comprising one or more nucleic acid sequences of said plurality of parental nucleic acid molecules; and
(d) processing fetal nucleic acid sequence information from one or more fetal nucleic acid molecules against said parental nucleic acid sequence information to identify one or more variations in said one or more fetal nucleic acid molecules.
2 . The method of claim 1 , further comprising, prior to (a), generating a plurality of partitions comprising (i) a plurality of parental nucleic acid molecules derived from said parental biological sample; and (ii) said plurality of nucleic acid barcode molecules.
3 . The method of claim 1 , wherein in (c), said parental nucleic acid sequence information is generated by sequencing said barcoded parental nucleic acid molecules or derivatives thereof.
4 . The method of claim 1 , wherein, in (c), said parental nucleic acid sequence information comprises haplotype blocks, and wherein (d) comprises processing said fetal nucleic acid sequence information against at least a subset of said haplotype blocks.
5 . The method of claim 1 , further comprising obtaining, from a subject having a fetus, a maternal biological sample, and deriving from said maternal biological sample (i) said parental nucleic acid molecules; and (ii) fetal nucleic acid molecules of said fetus.
6 . The method of claim 5 , wherein said fetal nucleic acid molecules are derived from a cell-free portion of said maternal biological sample.
7 . The method of claim 5 , further comprising sequencing said fetal nucleic acid molecules or derivatives thereof to generate said fetal nucleic acid sequence information.
8 . The method of claim 5 , wherein said maternal biological sample is a blood sample from said subject.
9 . The method of claim 1 , wherein said one or more fetal nucleic acid molecules are from a fetus, and wherein said one or more fetal nucleic acid molecules are derived from a cell-free biological sample of a subject having said fetus.
10 . The method of claim 9 , wherein said cell-free biological sample is obtained from a bodily fluid of said subject.
11 . The method of claim 10 , wherein said bodily fluid is blood.
12 . The method of claim 10 , wherein said bodily fluid is serum.
13 . The method of claim 1 , further comprising enriching said one or more fetal nucleic acid molecules or derivatives thereof from a fetal sample.
14 . The method of claim 1 , wherein in (a) said parental nucleic acid molecules are derived from a maternal biological sample, and wherein, in (d), said parental nucleic acid sequence information comprises said one or more nucleic acid sequences derived from said maternal biological sample.
15 . The method of claim 14 , wherein said parental nucleic acid sequence information further comprises one or more nucleic acid sequences derived from a paternal biological sample.
16 . The method of claim 1 , wherein said plurality of partitions is a plurality of droplets.
17 . The method of claim 1 , wherein said plurality of partitions is a plurality of wells.
18 . The method of claim 1 , wherein said one or more variations comprise a copy number variation, an insertion, a deletion, a translocation, an inversion, a retrotransposon, a rearrangement, a repeat expansion, a duplication, a gene fusion, a single nucleotide polymorphisms, or any combination thereof.
19 . The method of claim 1 , wherein said a given partition of said plurality of partitions comprises a parental nucleic acid molecule, wherein said parental nucleic acid molecule has a length longer than 1 kb.
20 . The method of claim 19 , wherein said parental nucleic acid molecule has a length longer than 10 kb.
21 . The method of claim 1 , wherein said plurality of partitions further comprise a plurality of beads, wherein a bead of said plurality of beads comprises a plurality of nucleic acid barcode molecules attached thereto, wherein a partition of said plurality of partitions comprises said bead.
22 . The method of claim 21 , wherein said bead comprises a plurality of nucleic acid barcode molecules comprising a common barcode sequence.
23 . The method of claim 22 , wherein said common barcode sequence is different from common barcode sequences of other beads of said plurality of beads.
24 . The method of claim 21 , wherein said plurality of beads is a plurality of gel beads.
25 . The method of claim 21 , wherein said plurality of nucleic acid barcode molecules is releasably attached to said bead.
26 . The method of claim 25 , wherein said plurality of nucleic acid barcode molecules is released upon application of a chemical stimulus.
27 . The method of claim 26 , wherein said chemical stimulus is a reducing agent.
28 . The method of claim 26 , wherein said partition further comprises said chemical stimulus.
29 . The method of claim 25 , wherein said plurality of nucleic acid barcode molecules is released upon application of a thermal stimulus.
30 . The method of claim 25 , wherein said plurality of nucleic acid barcode molecules is released upon application of a photo-stimulus.