DETECTION AND DIGITAL QUANTITATION OF MULTIPLE TARGETS
This disclosure provides for devices, methods, and systems for performing a non-invasive prenatal testing (NIPT) digital assay upon generating at least a large number of counts per chromosome for a set of chromosomes present in a sample, where performing the NIPT digital assay can include: distributing nucleic acids of the sample and materials for an amplification reaction across a plurality of partitions; amplifying the nucleic acids with the materials, within the plurality of partitions; and generating counts per chromosome upon detecting signals from the plurality of partitions. The inventions enable processing of samples for NIPT digital analyses and/or other digital analyses involving other loci of interest, with unprecedented partitioning, reaction, readout, and analytical performance.
1 .- 21 . (canceled)
22 . A method for performing a digital assay, the method comprising:
(a) obtaining a sample comprising a plurality of target nucleic acid molecules;
(b) generating a plurality of partitions, wherein said plurality of partitions comprises at least 1 million partitions, wherein a partition of the plurality of partitions comprises:
(i) materials for an amplification reaction, wherein the materials for the amplification reaction comprise primers comprising locked nucleic acid (LNA); and
(ii) less than or equal to one target nucleic acid molecule of the plurality of target nucleic acid molecules;
(c) amplifying said plurality of target nucleic acid molecules within the plurality of partitions; and
(d) detecting signals from said plurality of partitions, thereby generating counts corresponding to the plurality of target nucleic acid molecules.
23 . The method of claim 22 , wherein the plurality of target nucleic acid molecules comprises sequences associated with detecting a minimal residual disease (MRD).
24 . The method of claim 22 , wherein the plurality of target nucleic acid molecules comprises single nucleotide polymorphism (SNP) alleles.
25 . The method of claim 22 , wherein the primers comprise 10-60% LNA.
26 . The method of claim 22 , wherein the primers comprise up to six LNA bases.
27 . The method of claim 22 , wherein at least 50,000 counts are generated for each target of the plurality of target nucleic acid molecules.
28 . The method of claim 22 , wherein at least 100,000 counts are generated for each target of the plurality of target nucleic acid molecules.
29 . The method of claim 22 , wherein a dead volume associated with processing of the sample is less than 5%.
30 . The method of claim 22 , wherein the sample comprise a volume of less than 50 microliters.
31 . The method of claim 22 , wherein the plurality of target nucleic acid molecules comprise sequences associated with non-invasive prenatal testing (NIPT).
32 . The method of claim 22 , wherein said plurality of target nucleic acid molecules comprises sequences associated with a plurality of chromosomes, and wherein the sample comprises a maternal sample having a fetal fraction less than 6%.
33 . The method of claim 22 , wherein the plurality of target nucleic acid molecules comprise sequences associated with at least 3 chromosomes.
34 . The method of claim 22 , further comprising, after (b) and before (c), depositing the plurality of partitions into a well of a well plate.
35 . The method of claim 22 , wherein the plurality of partitions comprises droplets.
36 . A method for performing a digital assay, the method comprising:
(a) obtaining a sample comprising a plurality of target nucleic acid molecules associated with minimal residual disease (MRD) detection;
(b) generating a plurality of partitions, wherein said plurality of partitions comprises at least 1 million partitions, wherein a partition of the plurality of partitions comprises:
(i) materials for an amplification reaction; and
(ii) less than or equal to one target nucleic acid molecule of the plurality of target nucleic acid molecules;
(c) amplifying said plurality of target nucleic acid molecules within the plurality of partitions; and
(d) detecting signals from said plurality of partitions, thereby generating counts corresponding to the plurality of target nucleic acid molecules.
37 . A method for performing a digital assay, the method comprising:
(a) obtaining a sample comprising a plurality of target nucleic acid molecules, wherein the plurality of target nucleic molecules comprises single nucleotide polymorphism (SNP) targets;
(b) generating a plurality of partitions, wherein said plurality of partitions comprises at least 1 million partitions, wherein a partition of the plurality of partitions comprises:
(i) materials for an amplification reaction, wherein the materials comprise a primer comprising locked nucleic acid (LNA); and
(ii) less than or equal to one target nucleic acid molecule of the plurality of target nucleic acid molecules;
(c) amplifying said plurality of target nucleic acid molecules within the plurality of partitions; and
(d) detecting signals from said plurality of partitions, thereby generating counts corresponding to the plurality of target nucleic acid molecules.
38 . The method of claim 37 , wherein the primers comprise 10-60% LNA bases.
39 . The method of claim 37 , wherein a dead volume associated with processing of the sample is less than 5%.
40 . The method of claim 37 , wherein the sample comprise a volume of less than 50 microliters.