Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of 66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine Correspondence should be addressed to K.Z. (E-mail: kzhang@bioeng.ucsd.edu) or Y.G. (E-mail: ygao@vcu.edu). Accession numbers. All sequence reads and methylation data have been deposited at GEO, with accession number GSE15007.Note: Supplementary information is available on the Nature Biotechnology website. AUTHOR CONTRIBUTIONS NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.DNA methylation is a primary epigenetic mechanism for transcriptional regulation during normal development and goes awry in many diseases, including cancers. Genome-scale patterns of DNA methylation have been characterized by microarray hybridization or bisulfite sequencing 1 . Microarray methods have enabled methylation to be quantified at 1,536 discrete CpG sites in the human genome with the GoldenGate assay 2,3 . They have also been coupled with methylated DNA immunoprecipitation or methyl-specific restriction enzyme digestion to quantify relative levels of DNA methylation, although the read-outs of such approaches are only averages of the levels of methylation of multiple adjacent CpG sites [4][5][6] .More recently, next-generation sequencing has enabled absolute quantification of DNA methylation with single-nucleotide resolution on a larger scale than previously possible. These efforts include bisulfite sequencing of PCR amplicons from human tissues and cancer cell lines 7-9 , single-molecule sequencing of reduced representation libraries from mouse embryonic stem cells 10,11 and whole-genome bisulfite sequencing of Arabidopsis thaliana 12,13 . Although whole-genome bisulfite sequencing of a mammalian genome should be technically feasible, the large genome sizes pose a considerable challenge 14 .Selection or enrichment of genomic targets prior to sequencing would substantially reduce sequencing cost. PCR-based target selection is highly specific, but cannot be multiplexed easily for...

In diploid mammalian genomes, parental alleles can exhibit different methylation patterns (allele-specific DNA methylation, ASM), which have been documented in a small number of cases except for the imprinted regions and X chromosomes in females. We carried out a chromosome-wide survey of ASM across 16 human pluripotent and adult cell lines using Illumina bisulfite sequencing. We applied the principle of linkage disequilibrium (LD) analysis to characterize the correlation of methylation between adjacent CpG sites on single DNA molecules, and also investigated the correlation between CpG methylation and single nucleotide polymorphisms (SNPs). We observed ASM on 23%~37% heterozygous SNPs in any given cell line. ASM is often cell-type-specific. Furthermore, we found that a significant fraction (38%~88%) of ASM regions is dependent on the presence of heterozygous SNPs in CpG dinucleotides that disrupt their methylation potential. This study identified distinct types of ASM across many cell types and suggests a potential role for CpG-SNP in connecting genetic variation with the epigenome.

Entrectinib is a fi rst-in-class pan-TRK kinase inhibitor currently undergoing clinical testing in colorectal cancer and other tumor types. A patient with metastatic colorectal cancer harboring an LMNA-NTRK1 rearrangement displayed a remarkable response to treatment with entrectinib, which was followed by the emergence of resistance. To characterize the molecular bases of the patient's relapse, circulating tumor DNA (ctDNA) was collected longitudinally during treatment, and a tissue biopsy, obtained before entrectinib treatment, was transplanted in mice (xenopatient), which then received the same entrectinib regimen until resistance developed. Genetic profi ling of ctDNA and xenopatient samples showed acquisition of two point mutations in the catalytic domain of NTRK1 , p.G595R and p.G667C. Biochemical and pharmacologic analysis in multiple preclinical models confi rmed that either mutation renders the TRKA kinase insensitive to entrectinib. These fi ndings can be immediately exploited to design next-generation TRKA inhibitors. SIGNIFICANCE:We provide proof of principle that analyses of xenopatients (avatar) and liquid biopsies allow the identifi cation of drug resistance mechanisms in parallel with clinical treatment of an individual patient. We describe for the fi rst time that p.G595R and p.G667C TRKA mutations drive acquired resistance to entrectinib in colorectal cancers carrying NTRK1 rearrangements. Cancer Discov; 6(1);[36][37][38][39][40][41][42][43][44]

Here, we describe the dramatic response of a patient with an ETV6-NTRK3-driven mammary analogue secretory carcinoma to treatment with a pan-Trk inhibitor, and the development of acquired resistance linked to a novel NTRK3 mutation that interferes with drug binding. This case emphasizes how molecular profiling can identify therapies for rare diseases and dissect mechanisms of drug resistance.

ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.