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 - 120 . (canceled)
121 . A method of processing nucleic acid molecules, comprising:
(a) providing a plurality of nucleic acid molecules, wherein said plurality of nucleic acid molecules comprises less than about 50 nanograms (ng) of nucleic acid molecules;
(b) partitioning said plurality of nucleic acid molecules with a plurality of beads into a plurality of partitions, wherein a partition of said plurality of partitions comprises a nucleic acid molecule of said plurality of nucleic acid molecules and a bead of said plurality of beads, wherein said bead comprises a plurality of oligonucleotides coupled thereto, wherein an oligonucleotide of said plurality of oligonucleotides comprises a barcode sequence; and;
(c) within said partition:
(i) subjecting said bead to conditions sufficient to release said oligonucleotide from said bead; and
(ii) using said oligonucleotide released from said bead and said nucleic acid molecule to generate a nucleic acid product, wherein said nucleic acid product comprises said barcode sequence, or complement thereof, and a sequence of said nucleic acid molecule, or complement thereof.
122 . The method of claim 121 , further comprising recovering said nucleic acid product, or derivative thereof, from said partition.
123 . The method of claim 121 , further comprising identifying (i) said barcode sequence, or complement thereof, and (ii) said sequence of said nucleic acid molecule.
124 . The method of claim 123 , further comprising detecting a single nucleotide polymorphism, an insertion, or a deletion of said nucleic acid molecule, wherein said nucleic acid molecule comprises genomic deoxyribonucleic acid.
125 . The method of claim 123 , wherein the sensitivity for insertions and deletions is at least 70%, the positive predictive value of insertions and deletions is at least 90%, the sensitivity of single nucleotide polymorphisms is at least 95%, or the positive predictive value of single nucleotide polymorphisms is at least 95%.
126 . The method of claim 121 , wherein subjecting said bead to conditions sufficient to release said oligonucleotide from said bead comprises subjecting said bead to conditions sufficient to at least partially degrade said bead.
127 . The method of claim 121 , wherein subjecting said bead to conditions sufficient to release said oligonucleotide from said bead comprises applying a stimulus to said bead.
128 . The method of claim 127 , wherein said stimulus comprises a temperature change, a pH change, light, a chemical species, a reducing agent, or any combination thereof.
129 . The method of claim 121 , wherein said plurality of nucleic acid molecules comprises less than about 20 ng of nucleic acid molecules.
130 . The method of claim 129 , wherein said plurality of nucleic acid molecules comprises less than about 1 ng of nucleic acid molecules.
131 . The method of claim 121 , wherein said oligonucleotide comprises a primer sequence.
132 . The method of claim 131 , wherein said primer sequence comprises a random N-mer sequence.
133 . The method of claim 131 , wherein said primer sequence comprises a targeted primer sequence.
134 . The method of claim 121 , wherein (c) comprises performing a primer extension reaction.
135 . The method of claim 121 , wherein (c) comprises performing a ligation reaction.
136 . The method of claim 121 , wherein said barcode sequence comprises 6-20 nucleotides.
137 . The method of claim 121 , wherein said plurality of partitions is a plurality of wells.
138 . The method of claim 121 , wherein said plurality of partitions is a plurality of droplets.
139 . The method of claim 121 , wherein said plurality of nucleic acid molecules is derived from a bodily fluid.
140 . The method of claim 121 , wherein said plurality of nucleic acid molecules comprises nucleic acid molecules derived from circulating tumor nucleic acid.
141 . The method of claim 121 , wherein said plurality of nucleic acid molecules comprises nucleic acid molecules derived from fetal nucleic acid.