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 first nucleic acid target and a second nucleic acid target in a sample, the method comprising:
 (a) providing a mixture comprising:
 (i) a first plurality of nucleic acid molecules derived from, and/or corresponding with, said first nucleic acid target; 
 (ii) a second plurality of nucleic acid molecules derived from, and/or corresponding with said second nucleic acid target; 
 (iii) a first plurality of oligonucleotide probes, each of which is substantially complementary or homologous to a region of said first nucleic acid target; and 
 (iv) a second plurality of oligonucleotide probes, each of which is substantially complementary or homologous to a region of said second nucleic acid target; 
   (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;   (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 first nucleic acid target and said second nucleic acid target in said sample wherein such quantification only uses partitions positive for zero or one of the targets and no determined number of partitions positive for two or more of said 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 said first nucleic acid target or second nucleic acid target 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 said first nucleic acid target comprises a first chromosome and said second nucleic acid target comprises a second chromosome. 
     
     
         9 . The method of  claim 1 , wherein said first nucleic acid target or second nucleic target is a genomic region. 
     
     
         10 . The method of  claim 1 , wherein said first nucleic acid target or second nucleic target is derived from a virus or bacteria. 
     
     
         11 . The method of  claim 1 , wherein said first nucleic acid target or second nucleic target comprises RNA. 
     
     
         12 . The method of  claim 1 , wherein said first nucleic acid target or second nucleic target comprises nucleic acids derived from a fetus. 
     
     
         13 . The method of  claim 1 , wherein said first nucleic acid target is derived from a first organism and said second nucleic target comprises nucleic acids is derived from a second organism. 
     
     
         14 . The method of  claim 1 , wherein said first nucleic acid target is derived from a first individual and said second nucleic target 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 first nucleic acid target or said second nucleic acid target. 
     
     
         20 . The method of  claim 1 , wherein said quantifying comprises an relative quantification of said first nucleic acid target or said second 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 the first or second plurality of nucleic acid 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 the first or second plurality of nucleic acid molecules 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.