Methods for quantitative genetic analysis of cell free dna

Abstract

The invention provides a method for genetic analysis in individuals that reveals both the genetic sequences and chromosomal copy number of targeted and specific genomic loci in a single assay. The present invention further provides methods for the sensitive and specific detection of target gene sequences and gene expression profiles.

1 . A method for genetic analysis of cell-free DNA (cfDNA) comprising:
 (a) treating cfDNA with one or more end-repair enzymes to generate end-repaired cfDNA;   (b) ligating one or more adaptors to each end of the end-repaired cfDNA to generate a cfDNA library;   (c) amplifying the cfDNA library to generate cfDNA library clones;   (d) determining the number of genome equivalents in the cfDNA clone library; and   (e) performing a quantitative genetic analysis of one or more target genetic loci in the cfDNA library clones.   
     
     
         2 . The method of  claim 1 , further comprising isolating cfDNA from a biological sample of a subject. 
     
     
         3 . The method of  claim 1 , wherein the cfDNA is isolated from a biological sample selected from the group consisting of: amniotic fluid, blood, plasma, serum, semen, lymphatic fluid, cerebral spinal fluid, ocular fluid, urine, saliva, stool, mucous, and sweat. 
     
     
         4 . The method of  claim 1 , wherein
 (a) the one or more adaptors comprise a plurality of adaptor species;   (b) the one or more adaptors each comprise a primer binding site for amplification of the cfDNA library;   (c) the one or more adaptors each comprise one or more unique read codes;   (d) the one or more adaptors each comprise one or more sample codes for sample multiplexing; or   (e) the one or more adaptors each comprise one or more sequences for DNA sequencing.   
     
     
         5 .- 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein qPCR is performed on the cfDNA clone library and a qPCR measurement is compared to standards of known genome equivalents to determine the genome equivalents of the cfDNA clone library. 
     
     
         10 . The method of  claim 9 , wherein the qPCR is performed with a primer that binds to an Alu sequence and a primer that binds to a sequence in an adaptor. 
     
     
         11 . The method of  claim 1 , wherein:
 (a) the quantitative genetic analysis is performed on a plurality of genetic loci in the cfDNA library clones;   (b) the quantitative genetic analysis is performed on a plurality of genetic loci in a plurality of cfDNA clone libraries;   (c) the quantitative genetic analysis comprises hybridizing one or more capture probes to a target genetic locus to form capture probe-cfDNA clone complexes;   (d) the quantitative genetic analysis comprises isolating the capture probe-cfDNA clone complexes;   (e) the quantitative genetic analysis comprises amplification of the cfDNA clone sequence in the isolated hybridized capture probe-cfDNA clone complexes; or   (f) the quantitative genetic analysis comprises DNA sequencing to generate a plurality of sequencing reads.   
     
     
         12 .- 16 . (canceled) 
     
     
         17 . The method of  11 , further comprising bioinformatic analysis of the plurality of sequencing reads. 
     
     
         18 . The method of  claim 1 , wherein bioinformatics analysis is used:
 (a) to quantify the number of genome equivalents analyzed in the cfDNA clone library;   (b) to detect genetic variants in a target genetic locus;   (c) to detect mutations within a target genetic locus;   (d) to detect genetic fusions within a target genetic locus; and   (e) to measure copy number fluctuations within a target genetic locus.   
     
     
         19 . The method of  claim 2 , wherein:
 (a) the subject does not have a genetic disease;   (b) the subject has not been diagnosed with a genetic disease; or   (c) the subject has been diagnosed with a genetic disease.   
     
     
         20 .- 21 . (canceled) 
     
     
         22 . The method of  claim 19 , wherein the quantitative genetic analysis is used to identify or detect one or more genetic lesions that cause or associated with the genetic disease. 
     
     
         23 . The method of  claim 22 , wherein the genetic lesion comprises a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, a change in copy number, or a gene fusion. 
     
     
         24 . The method of  claim 22 , wherein the genetic lesion comprises a genomic rearrangement that fuses the 3′ coding region of the ALK gene to another gene. 
     
     
         25 . The method of  claim 24 , wherein the 3′ coding region of the ALK gene is fused to the EML4 gene. 
     
     
         26 . The method of  claim 22 , wherein the genetic disease is cancer. 
     
     
         27 . The method of  claim 2 , wherein the subject is pregnant. 
     
     
         28 . The method of  claim 27 , wherein the quantitative genetic analysis is used to identify or detect one or more genetic variants or genetic lesions of one or more target genetic loci in fetal cfDNA. 
     
     
         29 . The method of  claim 2 , wherein the subject is a transplant recipient. 
     
     
         30 . The method of  claim 27 , wherein the quantitative genetic analysis is used to identify or detect donor cfDNA in the subject. 
     
     
         31 . A method of predicting, diagnosing, or monitoring a genetic disease in a subject comprising:
 (a) isolating or obtaining cfDNA from a biological sample of a subject;   (b) treating the cfDNA with one or more end-repair enzymes to generate end-repaired cfDNA;   (c) ligating one or more adaptors to each end of the end-repaired cfDNA to generate a cfDNA library;   (d) amplifying the cfDNA library to generate a cfDNA clone library;   (e) determining the number of genome equivalents in the cfDNA clone library; and   (f) performing a quantitative genetic analysis of one or more target genetic loci associated with the genetic disease in the cfDNA clone library, wherein the identification or detection of one or more genetic lesions in the one or more target genetic loci is prognostic for, diagnostic of, or monitors the progression of the genetic disease.   
     
     
         32 . The method of  claim 31 , wherein the cfDNA is isolated from a biological sample selected from the group consisting of: amniotic fluid, blood, plasma, serum, semen, lymphatic fluid, cerebral spinal fluid, ocular fluid, urine, saliva, stool, mucous, and sweat. 
     
     
         33 . The method of  claim 31 , wherein the genetic lesion comprises a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, a change in copy number, or a gene fusion. 
     
     
         34 . The method of  claim 31 , wherein the genetic lesion comprises a genomic rearrangement that fuses the 3′ coding region of the ALK gene to another gene. 
     
     
         35 . The method of  claim 32 , wherein the 3′ coding region of the ALK gene is fused to the EML4 gene. 
     
     
         36 . The method of  claim 31 , wherein the genetic disease is cancer. 
     
     
         37 . A companion diagnostic for a genetic disease comprising:
 (a) isolating or obtaining cfDNA from a biological sample of a subject;   (b) treating the cfDNA with one or more end-repair enzymes to generate end-repaired cfDNA;   (c) ligating one or more adaptors to each end of the end-repaired cfDNA to generate a cfDNA library;   (d) amplifying the cfDNA library to generate a cfDNA clone library;   (e) determining the number of genome equivalents in the cfDNA clone library; and   (f) performing a quantitative genetic analysis of one or more biomarkers associated with the genetic disease in the cfDNA clone library, wherein detection of, or failure to detect, at least one of the one or more biomarkers indicates whether the subject should be treated for the genetic disease.   
     
     
         38 . The method of  claim 37 , wherein the cfDNA is isolated from a biological sample selected from the group consisting of: amniotic fluid, blood, plasma, serum, semen, lymphatic fluid, cerebral spinal fluid, ocular fluid, urine, saliva, stool, mucous, and sweat. 
     
     
         39 . The method of  claim 37 , wherein the biomarker is a genetic lesion. 
     
     
         40 . The method of  claim 39 , wherein the genetic lesion comprises a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, a change in copy number, or a gene fusion. 
     
     
         41 . The method of  claim 39 , wherein the genetic lesion comprises a genomic rearrangement that fuses the 3′ coding region of the ALK gene to another gene. 
     
     
         42 . The method of  claim 41 , wherein the 3′ coding region of the ALK gene is fused to the EML4 gene. 
     
     
         43 . The method of  claim 37 , wherein the genetic disease is cancer.