The concentration of purified bisulfite converted selleck chemical Brefeldin A DNA was de termined by quantitative real time PCR using bisulfite conversion specific primers for ACTB. A total of 59 conversion specific PCRs across 27 genes in triplicate were ap plied to 5 10 ng bisulfite treated DNA including, periph eral blood lymphocyte DNA and a 1 1 mix of wbc DNA and enzymatically methylated DNA. The triplicates were pooled and the concentration of PCR products estimated by gel electrophoresis. Equivalent amounts of the above 59 amplicons derived from the same patient or control samples were pooled, resulting in 22 pools. A total of 500 ng of each DNA pool was ligated with a bar coded MID linker so that the sample of origin for each read could be de duced from the sequence.

Libraries of pooled ampli cons were prepared Inhibitors,Modulators,Libraries following protocols provided with the Roche Library Inhibitors,Modulators,Libraries Preparation Kit and reagents, except that Qiagen MinElute columns were used to remove excess MID linkers. The libraries were sequenced on two halves of a flow cell on the Roche 454 Titanium FLX system. one half contained all of the CRC samples Inhibitors,Modulators,Libraries and Inhibitors,Modulators,Libraries the other half the equivalently bar coded normal samples. Bisulfite sequencing reads were assigned Inhibitors,Modulators,Libraries to individual tissue samples using the bar code MID se quences and aligned against in silico bisulfite converted reference sequence with all C characters at CpG sites con verted to Y and C in all other contexts converted to T. After best alignment with SHRiMP V2. 04, the fraction of unconverted cytosines at each potential CpG methyla tion site was determined for each sample.

Samples from wbc DNA as well as a 1 1 mixture of methylated and wbc DNAs were analysed for quality control purposes. Quantitative assays for CHIR99021 FDA DNA methylation Methylation specific PCR assays and control cytosine free fragment assay were performed using primer pairs and assay conditions shown in Additional file 2 Table S4. Input levels of bisulfite treated DNA were quantified using by qPCR using the CFF assay and a standard curve of serially diluted human genomic DNA ranging from 100 ng to 100 pg. For each target fragment, amounts of methyl ated target DNA were quantified using a standard curve of fully methylated DNA, 40 pg, 200 pg, 1 ng and 5 ng mixed with unmethylated DNA to give a total of 5 ng DNA. The levels of methylated DNA of each sample were determined from the standard curve and combined with the amount input DNA to calculate the percentage methylation. Results Biomarker discovery strategy In order to identify DNA methylation biomarkers poten tially suitable for early diagnosis of colorectal cancer, we have combined different genome wide approaches as illustrated in Figure 1.