Generating cell-free dna libraries directly from blood

Abstract

The disclosure provides methods and kits for preparing sequencing library to detect chromosomal abnormality using cell-free DNA (cfDNA) without the need of first isolating the cfDNA from a liquid fraction of a test sample. In some embodiments, the method involves reducing the binding between the cfDNA and nucleosomal proteins without unwinding the cfDNA from the nucleosomal proteins. In some embodiments, the reduction of binding may be achieved by treating with a detergent or heating. In some embodiments, the method further involves freezing and thawing the test sample before reducing the binding between the cfDNA and the nucleosomal proteins. In some embodiments, the test sample is a peripheral blood sample from a pregnant woman including cfDNA of both a mother and a fetus. In other embodiments, the test sample is a peripheral blood sample from a patient known or suspected to have cancer.

What is claimed is: 
     
         1 . A kit for classifying a copy number variation in a fetal genome, said kit comprising:
 (a) a sample collection device for holding a maternal test sample comprising fetal and maternal nucleic acids;   (b) an in-process positive control (IPC) comprising one or more nucleic acids comprising one or more chromosomal aneuploidies of interest, wherein the IPC provides a qualitative positive sequence dose value for said one or more chromosomal aneuploidies of interest; and   (c) one or more fixatives for white blood cell nuclei, one or more nuclease inhibitors, one or more albumin depletion columns, one or more Ig depletion columns, one or more nonionic detergents or salts, or any combinations thereof.   
     
     
         2 . The kit of  claim 1 , wherein the one or more nonionic detergents comprise Tween-20. 
     
     
         3 . The kit of  claim 2 , wherein Tween-20 has concentrations from about 0.1% to about 5%. 
     
     
         4 . The kit of  claim 1 , wherein the one or more nucleic acids comprising one or more chromosomal aneuploidies of interest in the IPC comprise i) nucleic acids comprising one or more internal positive controls for calculating a first fetal fraction and detecting copy number variations at a first location on a reference genome; and ii) nucleic acids comprising one or more internal positive controls for calculating a second fetal fraction at a second location on the reference genome other than the first location on the reference genome for detecting the copy number variation in i). 
     
     
         5 . The kit of  claim 1 , wherein the IPC is configured to relate sequence information obtained for the maternal test sample to the sequence information obtained from a set of qualified samples that were sequenced at a different time. 
     
     
         6 . The kit of  claim 1 , wherein the IPC comprises markers to track one or more samples through a sequencing process. 
     
     
         7 . The kit of  claim 1 , further comprising one or more marker molecules comprising nucleic acids and/or nucleic acid mimics that provide one or more antigenomic marker sequences suitable for tracking and verifying sample integrity. 
     
     
         8 . The kit of  claim 7 , wherein said marker molecules comprise one or more mimetics selected from the group consisting of a morpholino derivative, a peptide nucleic acid (PNA), and a phosphorothioate DNA. 
     
     
         9 . The kit of  claim 1 , wherein said sample collection device comprises a device for collecting blood and, optionally a receptacle for containing blood. 
     
     
         10 . The kit of  claim 9 , wherein said device for collecting blood and/or said receptacle for containing blood comprises an anticoagulant and/or cell fixative, and/or said antigenomic marker sequence(s) and/or said IPC. 
     
     
         11 . The kit of  claim 1 , wherein said kit further comprises reagents for sequencing library preparation. 
     
     
         12 . The kit of  claim 11 , wherein said reagents comprise a solution for end-repairing DNA, and/or a solution for dA-tailing DNA, and/or a solution for adaptor ligating DNA. 
     
     
         13 . The kit of  claim 1 , further comprising a consumable portion of a sequencer, wherein the consumable portion is configured to sequence fetal and maternal nucleic acids from one or more maternal test samples. 
     
     
         14 . The kit of  claim 13 , wherein the consumable portion of the sequencer is a flow cell. 
     
     
         15 . The kit of  claim 13 , wherein the consumable portion of the sequencer is a chip configured to detect ions. 
     
     
         16 . The kit of  claim 1 , wherein the IPC comprises a trisomy selected from the group consisting of trisomy 21, trisomy 18, trisomy 21, trisomy 13, trisomy 16, trisomy 13, trisomy 9, trisomy 8, trisomy 22, XXX, XXY, and XYY. 
     
     
         17 . The kit of  claim 1 , wherein the IPC comprises a trisomy selected from the group consisting of trisomy 21 (T21), and/or a trisomy 18 (T18), and/or a trisomy 13 (T13). 
     
     
         18 . The kit of  claim 1 , wherein the IPC comprises trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13). 
     
     
         19 . The kit of  claim 1 , wherein the IPC comprises an amplification or a deletion of a p arm or a q arm of any one or more of chromosomes 1-22, X and Y. 
     
     
         20 . A kit for classifying a copy number variation in a cancer genome, said kit comprising:
 (a) a sample collection device for holding a cancer patient test sample comprising cancer and non-cancer nucleic acids;   (b) an in-process positive control (IPC) comprising one or more nucleic acids comprising one or more chromosomal aneuploidies of interest, wherein the IPC provides a qualitative positive sequence dose value for said one or more chromosomal aneuploidies of interest; and   (c) one or more fixatives for white blood cell nuclei, one or more nuclease inhibitors, one or more albumin depletion columns, one or more Ig depletion columns, one or more nonionic detergents or salts, or combinations thereof.