Noninvasive diagnostics by sequencing 5-hydroxymethylated cell-free dna

Abstract

Provided herein is a method of sequencing hydroxymethyated cell-free DNA. In some embodiments, the method comprises adding an affinity tag to only hydroxymethyated DNA molecules in a sample of cfDNA, enriching for the DNA molecules that are tagged with the affinity tag; and sequencing the enriched DNA molecules.

What is claimed is: 
     
         1 . A method comprising:
 (a) obtaining a sample comprising circulating cell-free DNA;   (b) enriching for the hydroxymethylated DNA in the sample; and   (c) independently quantifying the amount of nucleic acids in the enriched hydroxymethylated DNA that map to each of one or more target loci.   
     
     
         2 . The method of  claim 1 , further comprising:
 (d) determining whether one or more nucleic acid sequences in the enriched hydroxymethylated DNA are over-represented or under-represented in the enriched hydroxymethylated DNA.   
     
     
         3 . The method of  claim 2 , further comprising:
 (e) making a diagnosis, a treatment decision or a prognosis bases on results based on the identity of the nucleic acids that are over-represented or under-represented in the enriched hydroxymethylated DNA.   
     
     
         4 . The method of  claim 3 , wherein the diagnosis, treatment decision or prognosis, wherein the diagnosis, treatment decision or prognosis is a cancer diagnosis. 
     
     
         5 . The method of  claim 4 , wherein the target loci include one or more of the following gene bodies: ABRACL, ADAMTS4, AGFG2, ALDH1A3, ALG10B, AMOTL1, APCDD1L-AS1, ARL6IP6, ASF1B, ATP6V0A2, AUNIP, BAGE, C2orf62, C8orf22, CALCB, CC2D1B, CCDC33, CCNL2, CLDN15, COMMD6, CPLX2, CRP, CTRC, DACH1, DAZL, DDX11L1, DHRS3, DUSP26, DUSP28, EPN3, EPPIN-WFDC6, ETAA1, FAM96A, FENDRR, FLJ16779, FLJ31813, GBX1, GLP2R, GMCL1P1, GNPDA2, GPR26, GSTP1, HMOX2, HOXC5, IGSF9B, INSC, INSL4, IRF7, KIF16B, KIF20B, LARS, LDHD, LHX5, LINC00158, LINC00304, LOC100128946, LOC100131234, LOC100132287, LOC100506963, LOC100507250, LOC100507410, LOC255411, LOC729737, MAFF, NPAS4, NRADDP, P2RX2, PAIP1, PAX1, PODXL2, POU4F3, PSMG1, PTPN2, RAG1, RBM14-RBM4, RDH11, RFPL3, RNF122, RNF223, RNF34, SAMD11, SHISA2, SIGLEC10, SLAMF7, SLC25A46, SLC25A47, SLC9A3R2, SORD, SOX18, SPATA31E1, SSR2, STXBP3, SYT11, SYT2, TCEA3, THAP7-AS1, TMEM168, TMEM65, TMX2, TPM4, TPO, TRAM1, TTC24, UBQLN4, WASH7P, ZNF284, ZNF423, ZNF444, ZNF800, ZNF850, and ZRANB2. 
     
     
         6 . The method of  claim 4 , wherein the target loci include one or more of the following intervals in the hg19 reference genome: chr1:114670001-114672000, chr1:169422001-169424000, chr1:198222001-198224000, chr1:239846001-239848000, chr1:24806001-24808000, chr1:3234001-3236000, chr1:37824001-37826000, chr1:59248001-59250000, chr1:63972001-63974000, chr1:67584001-67586000, chr1:77664001-77666000, chr2:133888001-133890000, chr2:137676001-137678000, chr2:154460001-154462000, chr2:200922001-200924000, chr2:213134001-213136000, chr2:219148001-219150000, chr2:41780001-41782000, chr2:49900001-49902000, chr3:107894001-107896000, chr3:108506001-108508000, chr3:137070001-137072000, chr3:17352001-17354000, chr3:23318001-23320000, chr3:87312001-87314000, chr3:93728001-93730000, chr4:39342001-39344000, chr4:90790001-90792000, chr5:103492001-103494000, chr5:39530001-39532000, chr5:83076001-83078000, chr6:122406001-122408000, chr6:129198001-129200000, chr6:156800001-156802000, chr6:157286001-157288000, chr6:45304001-45306000, chr7:11020001-11022000, chr7:13364001-13366000, chr8:42934001-42936000, chr8:53686001-53688000, chr8:69672001-69674000, chr9:3496001-3498000 and chr9:88044001-88046000. 
     
     
         7 . The method of  claim 4  wherein cancer diagnosis includes an indication of the tissue-type of the cancer. 
     
     
         8 . The method of  claim 1 , wherein step (c) is done by sequencing, digital PCR or hybridization to an array. 
     
     
         9 . The method of  claim 1 , wherein the determining step is done relative to a control, wherein the control comprises one or more control sequences in:
 the enriched hydroxymethylated DNA;   the sample of (a);   the sample of (a), after the hydroxymethylated DNA has been removed in step (b); or   a different sample.