Assay systems for genetic analysis

Abstract

The present invention provides assay systems and methods for detection of copy number variation at one or more loci and polymorphism detection at one or more loci in a mixed sample from an individual.

What is claimed is: 
     
         1 . A method for determining a presence or absence of a copy number variation of a genomic region in a minor source for each of a plurality of mixed samples obtained from a plurality of different individuals, the mixed samples comprising major source and minor source cell-free genomic DNA, the method comprising:
 (a) obtaining major source and minor source cell-free genomic DNA from each of the plurality of mixed samples;   (b) creating contiguous ligation product templates from selected loci of the major source and minor source cell-free genomic DNA from the mixed samples, wherein the contiguous ligation product templates comprise a sample index that identifies the cell-free DNA as being from an individual mixed sample;   (c) pooling the contiguous ligation product templates to produce a pool of indexed DNA corresponding to a first chromosome and indexed DNA corresponding to at least a second chromosome for the plurality of mixed samples;   (d) sequencing the pool of the contiguous ligation product templates to produce sequence reads corresponding to selected loci of the major source and minor source cell-free genomic DNA;   (f) quantifying the sequence reads corresponding to the indexed major source and minor source loci from the first chromosome and the at least second chromosome from individual mixed samples based on the sample index and without aligning to a reference sequence; and   (g) determining the presence or absence of a copy number variation for individual mixed samples based on the quantity of sequence reads corresponding to the indexed major source and minor source loci from the first chromosome of interest and the quantity of sequence reads corresponding to the indexed major source and minor source loci from at least second chromosome of interest.   
     
     
         2 . The method of  claim 1 , wherein for each of the plurality of mixed samples, determining the presence or absence of a copy number variation comprises comparing the number of sequence reads corresponding to selected loci of the first chromosome with the number of sequence reads corresponding to selected loci of the second chromosome. 
     
     
         3 . The method of  claim 1 , wherein the selected loci are from 10 to 150 nucleotide bases in length. 
     
     
         4 . The method of  claim 3 , wherein the selected loci are from 50 to 150 nucleotide bases in length. 
     
     
         5 . The method of  claim 1 , wherein the first chromosome is chromosome 13, chromosome 18, chromosome 21, X, or Y. 
     
     
         6 . The method of  claim 1 , wherein the presence or absence of a copy number variation is determined for chromosome 13, chromosome 18, chromosome 21, X, or Y. 
     
     
         7 . The method of  claim 6 , wherein the copy number variation determined is trisomy 21, trisomy 18, trisomy 13 or monosomy X. 
     
     
         8 . The method of  claim 6 , wherein the first chromosome is chromosome 21, and the second chromosome is chromosome 18. 
     
     
         9 . The method of  claim 1 , wherein the contiguous ligation product templates are amplified prior to performing the sequencing. 
     
     
         10 . The method of  claim 1 , wherein the amplifying comprises performing polymerase chain reaction (PCR) amplification on the contiguous ligation product templates. 
     
     
         11 . The method of  claim 10 , wherein PCR amplification comprises hybridizing at least two oligonucleotides comprising universal primer sequences to contiguous ligation product templates from the first chromosome and contiguous ligation product templates from the at least second chromosome. 
     
     
         12 . The method of  claim 10 , wherein each of the oligonucleotides used in amplification has a substantially similar melting temperature. 
     
     
         13 . The method of  claim 9 , wherein amplification is performed before pooling and the sample index is added to the contiguous ligation product template during universal amplification. 
     
     
         14 . The method of  claim 1 , wherein the sample index is added in the creation of the contiguous ligation product templates. 
     
     
         15 . The method of  claim 1 , wherein the sequencing is massively parallel sequencing. 
     
     
         16 . The method of  claim 1 , wherein the sequence reads are assigned to expected assay structures without use of a reference sequence. 
     
     
         17 . A method for determining a presence or absence of a copy number variation in a minor source for each of a plurality of mixed samples obtained from a plurality of different individuals, the mixed samples comprising major source and minor source cell-free genomic DNA, the method comprising:
 (a) obtaining major source and minor source cell-free genomic DNA from each of the plurality of mixed samples;   (b) creating contiguous ligation product templates from selected loci of the major source and minor source cell-free genomic DNA from the mixed samples, wherein at least 100 loci are selected from a first chromosome and at least 100 loci are selected from at least a second chromosome in the major source and minor source cell-free genomic DNA, and wherein the contiguous ligation product templates comprise a sample index that identifies the cell-free DNA as being from an individual mixed sample;   (c) pooling the contiguous ligation product templates to produce a pool of indexed DNA corresponding to a first chromosome and indexed DNA corresponding to a second chromosome for the plurality of mixed samples;   (d) performing sequencing of the pool of the contiguous ligation product templates to produce sequence reads corresponding to the selected loci of the major source and minor source cell-free genomic DNA;   (f) quantifying the sequence reads corresponding to the indexed major source and minor source loci from the first chromosome and the at least second chromosome from individual mixed samples based on the sample index and without aligning to a reference sequence; and   (g) determining the presence or absence of a copy number variation for individual mixed samples based on the quantity of sequence reads corresponding to the indexed major source and minor source loci from the first chromosome of interest and the quantity of sequence reads corresponding to the indexed major source and minor source loci from second chromosome of interest.   
     
     
         18 . The method of  claim 17 , wherein for each of the plurality of mixed blood samples, determining the presence or absence of a copy number variation comprises comparing the number of sequence reads corresponding to selected loci of the first chromosome with the number of sequence reads corresponding to selected loci of the second chromosome. 
     
     
         19 . The method of  claim 17 , wherein the selected loci are from 10 to 150 nucleotide bases in length. 
     
     
         20 . The method of  claim 19 , wherein the selected loci are from 50 to 150 nucleotide bases in length. 
     
     
         21 . The method of  claim 17 , wherein the first chromosome is chromosome 13, chromosome 18, chromosome 21, X, or Y. 
     
     
         22 . The method of  claim 17 , wherein the presence or absence of a copy number variation is determined for chromosome 13, chromosome 18, chromosome 21, X, or Y. 
     
     
         23 . The method of  claim 22 , wherein the copy number variation determined is trisomy 21, trisomy 18, trisomy 13 or monosomy X. 
     
     
         24 . The method of  claim 22 , wherein the first chromosome is chromosome 21, and the second chromosome is chromosome 18. 
     
     
         25 . The method of  claim 17 , wherein the contiguous ligation product templates are amplified using universal amplification prior to performing massively parallel sequencing. 
     
     
         26 . The method of  claim 25 , wherein the amplifying comprises performing polymerase chain reaction (PCR) amplification on the contiguous ligation product templates. 
     
     
         27 . The method of  claim 26 , wherein PCR amplification comprises hybridizing at least two oligonucleotides comprising universal primer sequences to the at least 100 different contiguous ligation product templates from the first chromosome and at least 100 different contiguous ligation product templates from the at least second chromosome. 
     
     
         28 . The method of  claim 27 , wherein each of the oligonucleotides used in amplification has a substantially similar melting temperature. 
     
     
         29 . The method of  claim 25 , wherein universal amplification is performed before pooling and the sample index is added to the contiguous ligation product template during universal amplification. 
     
     
         30 . The method of  claim 17 , wherein the sequence reads are assigned to expected assay structures without use of a reference sequence.