METHODS AND COMPOSITIONS FOR LABELING TARGETS AND HAPLOTYPE PHASING
Disclosed herein are methods for estimating the copy number of a target chromosome in a sample. In some embodiments, the methods include: partitioning a sample comprising one or more copies of the first target chromosome, into a plurality of partitioned samples; stochastically barcoding the one or more copies of the first target chromosome in the plurality of partitioned samples using a first plurality of stochastic barcodes, wherein each of the first plurality of stochastic barcodes comprises a first chromosome label and a first molecular label; and estimating the copy number of the first target chromosome in the sample using the first chromosome label and the second molecular label. The disclosed methods can be used for haplotype phasing, aneuploidy determination, and DNA sequencing.
1 . A method for estimating copy number of a target chromosome in a sample, comprising:
providing a sample comprising one or more copies of a first target chromosome;
partitioning the sample into a plurality of partitioned samples, wherein each of at least 10% of the plurality of partitioned samples comprises one copy of the first target chromosome;
stochastically barcoding the one or more copies of the first target chromosome in the plurality of partitioned samples using a first plurality of stochastic barcodes, wherein each of the first plurality of stochastic barcodes comprises a first chromosome label and a first molecular label; and
estimating the copy number of the first target chromosome in the sample using the first chromosome label and the second molecular label.
2 . The method of claim 1 , wherein partitioning the sample comprises adjusting the volume of the sample to alter the concentration of the first target chromosome in the sample.
3 . The method of claim 1 , wherein partitioning the sample comprises adjusting the volume of the sample partitioned into each of the plurality of partitioned samples.
4 . The method of claim 1 , wherein each of at least 25% of the plurality of partitioned samples comprises one copy of the first target chromosome.
5 .- 7 . (canceled)
8 . The method of claim 1 , wherein each of the plurality of partitioned samples is introduced to a well of a plurality of wells of a substrate.
9 . The method of claim 1 , wherein each of the plurality of partitioned samples is a droplet in an emulsion.
10 . (canceled)
11 . The method of claim 1 , wherein stochastically barcoding the one or more copies of the first target chromosome in the plurality of partitioned samples comprises generating one or more copies of a stochastically barcoded first target chromosome.
12 . The method of claim 11 , wherein stochastically barcoding the one or more copies of the first target chromosome comprises generating an indexed library of the stochastically barcoded first target chromosome.
13 . The method of claim 1 , wherein stochastically barcoding the one or more copies of the first target chromosome comprises fragmenting the one or more copies of the first target chromosome to generate fragments of the first target chromosome.
14 . The method of claim 13 , wherein the fragments of the first target chromosome are at least 10 kilo bases (kb), 100 kb, or 1000 kb in length.
15 .- 16 . (canceled)
17 . The method of claim 13 , wherein the stochastically barcoded first target chromosome comprises stochastically barcoded fragments of the first target chromosome.
18 . The method of claim 1 , wherein the first plurality of the stochastic barcodes is associated with a solid support.
19 .- 20 . (canceled)
21 . The method of claim 18 , wherein the first chromosome labels of the first plurality of stochastic barcodes on the solid support are the same.
22 . The method of claim 1 , wherein the first chromosome label is about 5-20 nucleotides long.
23 .- 25 . (canceled)
26 . The method of claim 17 , wherein estimating the copy number of the first target chromosome in the sample comprises determining sequences of at least some of the stochastically barcoded fragments of the first target chromosome in the indexed library.
27 . (canceled)
28 . The method of claim 1 , wherein the one or more copies of the first target chromosome are inside or not inside one or more cells.
29 .- 32 . (canceled)
33 . The method of claim 1 ,
wherein the sample comprises one or more copies of a second target chromosome, and
wherein each of at least 10% of the plurality of partitioned samples comprises one copy of the second target chromosome,
the method further comprising:
stochastically barcoding one or more copies of the second target chromosome in the plurality of partitioned samples using a second plurality of stochastic barcodes, wherein each of the second plurality of stochastic barcodes comprises a second chromosome label and a second molecular label, and wherein the first chromosome labels of the first plurality of stochastic barcodes and the second chromosome labels of the second plurality of stochastic barcodes differ by at least one nucleotide; and
estimating the copy number of the second target chromosome in the sample using the second chromosome label and the second molecular label.
34 . The method of claim 1 ,
wherein the sample comprises one or more copies of each of n target chromosomes, wherein n is an integer greater than one, and
wherein, for each of the n target chromosomes, each of at least 10% of the plurality of partitioned samples comprises one copy of the n th target chromosome,
the method further comprising:
for each of the n target chromosomes in the plurality of partitioned samples, stochastically barcoding the one or more copies of the n h target chromosome using a n th plurality of stochastic barcodes,
wherein each of the n th plurality of stochastic barcodes comprises a n th chromosome label and a n th molecular label, and wherein the first chromosome labels of the first plurality of stochastic barcodes and the n th chromosome labels of the n th plurality of stochastic barcodes differ by at least one nucleotide; and
estimating the copy number of each of the plurality of n th target chromosomes in the sample using the n th chromosome label and the n th molecular label.
35 . (canceled)
36 . A method for haplotype phasing two or more gene targets on a target chromosome in a sample, comprising:
providing a sample comprising one or more copies of a target chromosome, wherein the target chromosome comprises two or more gene targets;
partitioning the sample into a plurality of partitioned samples, wherein each of at least 10% of the plurality of partitioned samples comprises one copy of the target chromosome;
stochastically barcoding the one or more copies of the target chromosome in the plurality of partitioned samples using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a chromosome label and a molecular label; and
determining the haplotype phasing of the two or more gene targets on the target chromosome in the sample using the chromosome label and the molecular label.
37 .- 51 . (canceled)
52 . The method of claim 36 , wherein at least two of the two or more gene targets are separated from one another on the target chromosome by at least 10 kb, at least 100 kb, or at least 1000 kb.
53 .- 54 . (canceled)
55 . A method for determining aneuploidy of one or more cells, comprising:
providing a sample comprising chromosomes from one or more cells;
partitioning the sample into a plurality of partitioned samples, wherein each of at least 10% of the plurality of partitioned samples comprises one copy of a first target chromosome;
stochastically barcoding the one or more copies of the first target chromosome in the plurality of partitioned samples using a first plurality of stochastic barcodes, wherein each of the first plurality of stochastic barcodes comprises a first chromosome label and a first molecular label; and
determining the aneuploidy of the one or more cells in the sample, wherein determining the aneuploidy of the one or more cells in the sample comprises determining the number of a first gene target on the first target chromosome using the first chromosome label and the first molecular label.
56 .- 67 . (canceled)
68 . The method of claim 55 , wherein the aneuploidy is a trisomy.
69 . The method of claim 68 , wherein the trisomy is an autosomal trisomy.
70 .- 71 . (canceled)
72 . A method for sequencing a first target chromosome in a sample, comprising:
providing a sample comprising one or more copies of a first target chromosome;
partitioning the sample into a plurality of partitioned samples, wherein each of at least 10% of the plurality of partitioned samples comprises one copy of the first target chromosome;
stochastically barcoding the one or more copies of the first target chromosome in the plurality of partitioned samples using a first plurality of stochastic barcodes, wherein each of the first plurality of stochastic barcodes comprises a first chromosome label and a first molecular label; and
obtaining sequence information of the first target chromosome using the first chromosome label and the first molecular label.
73 .- 91 . (canceled)