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 . 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 5 million partitions, and 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, wherein at least 50,000 counts are generated for each target of the plurality of target nucleic acid molecules.
2 . The method of claim 1 , wherein the plurality of target nucleic acid molecules comprise sequences associated with a genomic disease.
3 . The method of claim 1 , wherein the plurality of target nucleic acid molecules comprise sequences associated with a minimal residual disease.
4 . The method of claim 1 , wherein generating the plurality of partitions is performed within a closed container, wherein amplifying the plurality of target nucleic acid molecules is performed within the closed container, and wherein signals from the plurality of partitions are detected from within the closed container.
5 . The method of claim 1 , wherein the plurality of partitions is provided as a clear emulsion.
6 . The method of claim 1 , wherein the clear emulsion has clarity without refractive index matching between components of the clear emulsion.
7 . The method of claim 1 , wherein said plurality of target nucleic acid molecules comprises sequences associated with a plurality of chromosomes.
8 . The method of claim 7 , wherein the sample comprises a maternal sample having a fetal fraction less than 6%.
9 . The method of claim 1 , wherein the sample comprises nucleic acid molecules extracted from plasma.
10 . The method of claim 1 , wherein the sample comprises nucleic acid molecules extracted from blood.
11 . The method of claim 1 , wherein the plurality of target nucleic acid molecules comprises single nucleotide polymorphisms.
12 . The method of claim 1 , wherein (b) through (d) is completed within a duration of no more than 3 hours.
13 . The method of claim 1 , wherein the plurality of partitions comprises at least 20 million partitions.
14 . The method of claim 1 , wherein a dead volume associated with generating the plurality of partitions from the sample is less than 5% of an original volume of the sample.
15 . A method for performing a digital assay, the method comprising:
(a) generating a plurality of partitions within a closed container, from a sample comprising a plurality of target nucleic acid molecules, wherein the plurality of partitions comprises at least 10 million partitions, and wherein each partition of said plurality of partitions comprises:
(iii) materials for an amplification reaction; and
(iv) less than or equal to one target nucleic acid molecule of said plurality of target nucleic acid molecules;
(b) amplifying said plurality of target nucleic acid molecules within said plurality of partitions; and
(c) detecting signals from said plurality of partitions, thereby generating counts corresponding to said plurality of target nucleic acid molecules, with at least 99% sensitivity for detection of a target component of the plurality of target nucleic acid molecules.
16 . The method of claim 15 , wherein a dead volume associated with generating the plurality of partitions from the sample is less than 5% of an original volume of the sample.
17 . The method of claim 15 , wherein generating the plurality of partitions is performed within a closed container, wherein amplifying the plurality of target nucleic acid molecules is performed within the closed container, and wherein signals from the plurality of partitions are detected from within the closed container.
18 . The method of claim 15 , wherein the plurality of partitions is provided as a clear emulsion and wherein the clear emulsion has clarity without refractive index matching between components of the clear emulsion.
19 . The method of claim 15 , wherein the sample comprises nucleic acid molecules extracted from blood.
20 . The method of claim 15 , wherein the plurality of partitions are not provided using microwells.