Methods and systems for multiplex analysis

Abstract

The present disclosure provides methods and compositions for multiplex quantitation. In some aspects, methods and compositions are provided for quantitation of nucleic acid targets from a biological sample. The disclosed methods may be useful in identifying or detecting genetic abnormalities from a subject.

What is claimed is: 
     
         1 . A method of quantifying a nucleic acid target in a sample, the method comprising:
 (a) providing a mixture comprising:   i. a plurality of nucleic acid molecules derived from, and/or corresponding with, at least three nucleic acid targets;   ii. a plurality of oligonucleotide probes, each of which is substantially complementary or homologous to regions of said at least three nucleic acid targets; and   (b) partitioning said mixture into a plurality of partitions;   (c) generating a plurality of signals in said plurality of partitions, wherein said plurality of signals are detectable in at least one color channel, and wherein a signal generated from at least one nucleic acid target being present is ambiguous with a signal generated from two or more other targets;   (d) detecting from multiple partitions of said plurality of partitions said plurality of signals in said color channel; and   (e) based on said detecting, quantifying said nucleic acid target in said sample wherein such quantification does not use a determination of number of partitions positive for said ambiguous signal generated from said two or more other targets.   
     
     
         2 . The method of  claim 1 , wherein at least a portion of said plurality of signals is generated by said first or second plurality of oligonucleotide probes. 
     
     
         3 . The method of  claim 1 , wherein said sample is derived from a biological sample. 
     
     
         4 . The method of  claim 3 , wherein said biological sample is blood or plasma. 
     
     
         5 . The method of  claim 3 , wherein said biological sample is a maternal plasma sample. 
     
     
         6 . The method of  claim 1 , wherein one or more nucleic acid targets of said at least three nucleic acid targets comprises a chromosome. 
     
     
         7 . The method of  claim 6 , wherein said chromosome is chromosome 21, chromosome 18, chromosome 15, chromosome 13, an X chromosome, or a Y chromosome. 
     
     
         8 . The method of  claim 6 , wherein a first nucleic acid target of said at least three nucleic acid targets comprises a first chromosome and a second nucleic acid target of said at least three nucleic acid targets comprises a second chromosome. 
     
     
         9 . The method of  claim 1 , wherein one or more nucleic acid targets of said at least three nucleic acid targets comprises is a genomic region. 
     
     
         10 . The method of  claim 1 , wherein one or more nucleic acid targets of said at least three nucleic acid targets comprises is derived from a virus or bacteria. 
     
     
         11 . The method of  claim 1 , wherein one or more nucleic acid targets of said at least three nucleic acid targets comprises RNA. 
     
     
         12 . The method of  claim 1 , wherein one or more nucleic acid targets of said at least three nucleic acid targets comprises nucleic acids derived from a fetus. 
     
     
         13 . The method of  claim 1 , wherein a first nucleic acid of said at least three nucleic acid targets is derived from a first organism and said second nucleic target of said at least three nucleic acid targets comprises nucleic acids is derived from a second organism. 
     
     
         14 . The method of  claim 1 , wherein a first nucleic acid of said at least three nucleic acid targets is derived from a first individual and said second nucleic target of said at least three nucleic acid targets comprises nucleic acids is derived from a second individual. 
     
     
         15 . The method of  claim 1 , wherein (c) comprises amplifying said plurality of nucleic acid molecules, thereby generating said plurality of signals. 
     
     
         16 . The method of  claim 15 , wherein said amplifying degrades said plurality of oligonucleotide probes, thereby generating said plurality of signals. 
     
     
         17 . The method of  claim 1 , wherein said plurality of oligonucleotide probes are degraded by a nucleic acid enzyme, thereby releasing a signal tag from each of said plurality of oligonucleotide probes and generating said plurality of signals. 
     
     
         18 . The method of  claim 1 , wherein said plurality of signals is a plurality of fluorescent signals or a plurality of chemiluminescent signals. 
     
     
         19 . The method of  claim 1 , wherein said quantifying comprises an absolute quantification of said nucleic acid target. 
     
     
         20 . The method of  claim 1 , wherein said quantifying comprises a relative quantification of said nucleic acid target. 
     
     
         21 . The method of  claim 1 , wherein said quantifying comprises generating an average number of nucleic acid molecules per partition. 
     
     
         22 . The method of  claim 1 , wherein said quantifying comprises generating a probability that one or more nucleic acid molecules of said nucleic acid target molecules is present in a given partition. 
     
     
         23 . The method of  claim 1 , wherein said quantifying further comprises, for said plurality of partitions, generating a probability that one or more nucleic acid molecules of said nucleic acid target is present in a given partition of said plurality of partitions, thereby generating a plurality of probabilities. 
     
     
         24 . The method of  claim 23 , wherein said quantifying further comprises summing two or more probabilities of said plurality of probabilities.