COMPOSITIONS AND METHODS FOR DIGITAL POLYMERASE CHAIN REACTION
In some aspects, the present disclosure provides methods for identifying sequence variants in a nucleic acid sample. In some embodiments, a method comprises distinguishing between a true mutation in a polynucleotide and a random error introduced during an amplification step. In some embodiments, the methods reduce the number of false positives reported by a digital PCR assay. In some embodiments, the methods improve the accuracy of a digital PCR assay.
1 . A method of identifying a sequence variant in a nucleic acid sample comprising a plurality of polynucleotides, said method comprising:
(a) circularizing said plurality of polynucleotides to form a plurality of circularized polynucleotides;
(b) amplifying said plurality of circularized polynucleotides to generate a plurality of concatemers, each comprising a plurality of sequence repeats;
(c) partitioning said plurality of concatemers into a plurality of partitions, such that, on average, no more than one concatemer comprising a target sequence is present in an individual partition,
wherein said individual partition of said plurality of partitions contains at least one of a first probe and a second probe, wherein said first probe binds to said target sequence that lacks said sequence variant and produces a first signal, and said second probe binds to said target sequence that contains said sequence variant and produces a second signal;
(d) detecting said first signal and said second signal from said individual partition; and
(e) identifying said sequence variant as present in said target sequence only when a level of said second signal exceeds that of a threshold level indicative of one copy of a target sequence, and a level of said first signal is below that of a threshold level indicative of one copy of a target sequence.
2 . The method of claim 1 , further comprising, identifying said sequence variant as absent when a level of said first signal exceeds that of a threshold level indicative of one copy of a target sequence and a level of said second signal is below that of a threshold level indicative of one copy of a target sequence.
3 . The method of claim 1 , further comprising, identifying a false positive when a level of said first signal exceeds that of a threshold level indicative of one copy of a target sequence, and a level of said second signal exceeds that of a threshold level indicative of one copy of a target sequence.
4 .- 5 . (canceled)
6 . The method of claim 1 , wherein said plurality of polynucleotides comprise single-stranded polynucleotides.
7 . The method of claim 1 , wherein said plurality of polynucleotides comprise cell-free DNA.
8 . The method of claim 1 , wherein said circularizing comprises ligating a 5′ end and a 3′ end of at least one of said plurality of polynucleotides.
9 . The method of any onc of claim 1 , wherein said circularizing comprises ligating an adapter to the 5′ end, the 3′ end, or both the 5′ end and the 3′ end of at least one of said plurality of polynucleotides.
10 . The method of any onc of claim 1 , wherein said amplifying comprises amplifying using a polymerase having strand-displacement activity.
11 . (canceled)
12 . The method of claim 1 , wherein said amplifying comprises subjecting said plurality of circular polynucleotides to an amplification reaction mixture comprising random primers.
13 . The method of claim 1 , wherein said amplifying comprises subjecting said plurality of circular polynucleotides to an amplification reaction mixture comprising one or more primers, each of which specifically hybridizes to a different target sequence via sequence complementarity.
14 . (canceled)
15 . The method of claim 1 , further comprising, prior to said partitioning, fragmenting said plurality of concatemers to generate a plurality of fragmented concatemers.
16 . The method of claim 15 , further comprising, after said fragmenting and prior to said partitioning, selecting a plurality of said fragmented concatemers based on size.
17 . The method of claim 1 , wherein said plurality of partitions comprise emulsion-based droplets.
18 . (canceled)
19 . The method of claim 1 , wherein said plurality of partitions comprise a well or a tube.
20 . The method of claim 1 , wherein said first probe comprises a first detectable label and said second probe comprises a second detectable label.
21 .- 24 . (canceled)
25 . The method of claim 1 , wherein said first probe and said second probe are Taqman assay-based probes.
26 . The method of claim 25 , further comprising, after said partitioning and before said detecting, performing a polymerase chain reaction on said concatemers to amplify a region of said plurality of sequence repeats.
27 . A method for reducing error in a digital polymerase chain reaction on a nucleic acid sample comprising less than 50 ng of polynucleotides, said method comprising:
(a) circularizing individual polynucleotides in said nucleic acid sample to generate a plurality of circularized polynucleotides;
(b) amplifying said plurality of circularized polynucleotides to form a plurality of concatemers, each comprising a plurality of sequence repeats;
(c) partitioning said plurality of concatemers into a plurality of partitions, such that, on average, no more than one concatemer comprising a target sequence is present in an individual partition,
wherein an individual partition of said plurality of partitions contains at least one of a first probe and a second probe, wherein said first probe binds to said plurality of sequence repeats that lack said sequence variant and produces a first signal, and said second probe binds to said plurality of sequence repeats that contain said sequence variant and produces a second signal;
(d) detecting said first signal and said second signal from said individual partition; and
(e) identifying a false positive when a level of said first signal exceeds that of a threshold level indicative of one copy of a target sequence, and a level of said second signal exceeds that of a threshold level indicative of one copy of a target sequence.
28 .- 29 . (canceled)
30 . The method of claim 27 , wherein said method reduces false positives by at least 20%.
31 . The method of claim 27 , wherein said nucleic acid sample comprises cell-free polynucleotides.
32 .- 41 . (canceled)