Methods and Compositions for Sample Analysis
The present disclosure relates to methods and systems for sample processing and analyzing when the total quantity of input sample is low or when a target of interest is present as a relatively minor or rare population within the overall sample. The disclosure particularly relates to analyzing nucleic acid samples, including samples where a target nucleic acid of interest is present as a relatively low proportion of the overall nucleic acids.
1 . A method of analyzing nucleic acids, comprising:
(a) providing a collection of nucleic acids derived from a nucleic acid sample, wherein the collection of nucleic acids includes nucleic acid molecules at an amount of less than 50 nanograms (ng);
(b) combining the collection of nucleic acids with a plurality of oligonucleotides releasably connected to beads to form a mixture;
(c) partitioning the mixture into a plurality of partitions and releasing the oligonucleotides from the beads within the partitions;
(d) amplifying the collection of nucleic acids within the partitions to form amplification products of the collection of nucleic acids;
(e) pooling the collection of nucleic acids and the amplification products to form a pooled mixture; and
(f) detecting nucleic acid sequences of at least a portion of nucleic acids within the pooled mixture.
2 . The method of claim 1 , wherein, in (f), the detecting is completed at an accuracy greater than 90%.
3 . The method of claim 2 , wherein, in (f), the detecting is completed at an accuracy greater than 95%.
4 . The method of claim 3 , wherein, in (f), the detecting is completed at an accuracy greater than 99%.
5 . The method of claim 1 , wherein, in (f), the detecting comprises detecting at least 90% of the nucleic acids within the collection of nucleic acids.
6 . The method of claim 1 , wherein, in (f), the detecting comprises detecting sequences of a minor population within the collection of nucleic acids, which minor population makes up less than 50% of the collection of nucleic acids.
7 . The method of claim 6 , wherein the minor population makes up less than 25% of the collection of nucleic acids.
8 . The method of claim 7 , wherein the minor population makes up less than 10% of the collection of nucleic acids.
9 . The method of claim 8 , wherein the minor population makes up less than 5% of the collection of nucleic acids.
10 . The method of claim 1 , wherein the amount is less than 40 ng.
11 . The method of claim 10 , wherein the amount is less than 20 ng.
12 . The method of claim 11 , wherein the amount is less than 10 ng.
13 . The method of claim 12 , wherein the amount is less than 5 ng.
14 . The method of claim 13 , wherein the amount is less than 1 ng.
15 . The method of claim 14 , wherein the amount is less than 0.1 ng.
16 . The method of claim 1 , wherein each of the plurality of oligonucleotides comprises at least a constant region and a variable region.
17 . The method of claim 16 , wherein the constant region comprises a barcode sequence.
18 . The method of claim 17 , wherein the barcode sequence is between about 6 nucleotides and about 20 nucleotides in length.
19 . The method of claim 16 , wherein the variable region comprises a primer sequence.
20 . The method of claim 19 , wherein, in (d), the plurality of oligonucleotides function as primers in amplifying the collection of nucleic acids.
21 . The method of claim 1 , wherein the oligonucleotides are released from the beads upon exposure to one or more stimuli.
22 . The method of claim 21 , wherein the stimuli comprise temperature, pH, light, chemical species, and/or reducing agent.
23 . The method of claim 22 , wherein the stimuli comprises a reducing agent that comprises dithiothreitol (DTT) or tris(2-carboxylethyl)phosphine (TCEP).
24 . The method of claim 1 , wherein the partitions comprise droplets, microcapsules, wells or tubes.
25 . The method of claim 1 , wherein the partitions are fluid droplets.
26 . The method of claim 25 , wherein the fluid droplets are aqueous droplets within a water-in-oil emulsion.
27 . The method of claim 1 , wherein, in (c), the partitions are generated by a microfluidic device.
28 . The method of claim 1 , wherein the collection of nucleic acids is derived from a bodily fluid.
29 . The method of claim 28 , wherein the bodily fluid comprises blood, plasma, serum, or urine.
30 . The method of claim 28 , wherein at least a subset of the collection of nucleic acids is derived from one or more circulating tumor cells.
31 . The method of claim 28 or 30 , wherein a subset of the nucleic acids are derived from a tumor.
32 . The method of claim 1 , wherein the collection of nucleic acids is derived from a tissue biopsy.
33 . The method of claim 1 , wherein the collection of nucleic acids comprises fetal nucleic acids.
34 . The method of claim 33 , wherein less than 5% of nucleic acids of the collection of nucleic acids comprises fetal nucleic acids.
35 . The method of claim 1 , wherein the nucleic acid sample comprises a cellular sample.
36 . The method of claim 35 , wherein the cellular sample comprises less than 5% circulating tumor cells.
37 . The method of claim 35 , wherein the cellular sample comprises less than 5% tumor cells.
38 . The method of claim 1 , wherein the nucleic acid sample is derived from a sample selected from the group consisting of a live sample, a non-conserved sample, a preserved sample, an embalmed sample and a fixed sample.
39 . The method of claim 38 , wherein the sample is an embedded sample.
40 . The method of claim 39 , wherein the sample is a formaldehyde fixed and paraffin embedded sample.
41 . The method of claim 31 , wherein the one or more circulating tumor cells are obtained from a non-conserved sample or from a formaldehyde fixed and paraffin embedded sample.
42 . A method of analyzing nucleic acids, comprising:
a) combining a collection of nucleic acids derived from a nucleic acid sample with a plurality of oligonucleotides releasably connected to beads to form a mixture;
b) partitioning the mixture into a plurality of partitions;
c) releasing the oligonucleotides from the beads within the partitions;
d) amplifying the collection of nucleic acids within the partitions to form amplification products of the collection of nucleic acids;
e) pooling the collection of nucleic acids and the amplification products to form a pooled mixture; and
f) detecting nucleic acid sequences of a minor population within the collection of nucleic acids in the pooled mixture, which minor population makes up less than 50% of the collection of nucleic acids.
43 .- 63 . (canceled)
64 . A method of analyzing nucleic acids, comprising:
a) providing a collection of nucleic acids derived from a nucleic acid sample, wherein the collection of nucleic acids includes nucleic acid molecules at an amount of less than 50 nanograms (ng);
b) combining the collection of nucleic acids with a plurality of oligonucleotides to form a mixture, wherein each of the plurality of oligonucleotides comprises at least a constant region and a variable region, which constant region comprises a barcode sequence;
c) partitioning the mixture into a plurality of partitions and amplifying the collection of nucleic acids within the partitions to form amplification products of the collection of nucleic acids;
d) pooling the collection of nucleic acids and the amplification products to form a pooled mixture; and
e) detecting nucleic acid sequences of at least a portion of nucleic acids within the pooled mixture at a sensitivity of at least 90%.
65 .- 74 . (canceled)
75 . A method for analyzing a nucleic acid sequence, comprising:
a) providing partitions comprising nucleic acid molecules generated from a nucleic acid sample;
b) pooling the nucleic acid molecules from the partitions into a nucleic acid mixture;
c) subjecting the nucleic acid mixture to nucleic acid sequencing to generate sequencing reads comprising nucleic acid sequences of the nucleic acid molecules;
d) using a programmed computer processor to (i) analyze the sequencing reads and (ii) identify at least one contaminant read in the sequencing reads that is associated with a contaminant nucleic acid molecule in the nucleic acid mixture;
e) removing the contaminant read from the sequencing reads; and
f) generating a sequence of the nucleic acid sample from the sequencing reads with the contaminant read removed.
76 .- 120 . (canceled)