A wide range of DNA inputs from Jurkat cells were bisulfite-treated using a dedicated EpiTect Fast protocol and libraries were prepared using the QIAseq Methyl Library Kit and sequenced on a MiSeq instrument. Enough library to load onto a MiSeq was generated from as little as 100 pg with high mapping rates.
A comparison of the QIAseq Methyl Library Kit and Supplier Z/I was carried out. Libraries were constructed from 10 ng of DNA extracted from hepatic endothelial cells. The minimum possible input using chemistries from other suppliers was 35 ng. Libraries were sequenced on HiSeq with approximately 4x depth. A Library quality: Numbers shown are a percentage of reads (mapped, duplicated and mapped which passed the quality filter and used for methylation analysis). Higher quality reads were generated with the QIAseq kit even with one-third of input DNA. B Methylation calls: High conversion rate during bisulfite treatment and library preparation led to accurate methylation calls. This is represented by the high, unbiased conversion rate of cytosine in a non-CpG context, 0.1% methylation of CHH and 0.8% methylation of CHG (CHH and CHG are considered unmethylated in mammalian cells). C GC bias: Low GC bias (35–85% GC content) was demonstrated using the QIAseq Methyl Library Kit.
The kit contains all reagents required to go from bisulfite-treated DNA samples to sequencing-ready libraries. The double strand synthesis, end polishing and adapter ligation steps happen in a
ccfDNA from three different healthy donors was isolated using the QIAamp MinElute ccfDNA Kit. Total eluates in the range of 8–10 ng were bisulfite- treated using DNA Protect and an optimized incubation cycle to ensure complete conversion and avoid further fragmentation. The complete bisulfite-treated DNA was used to generate libraries using the QIAseq Methyl Library Kit. Quality control of the libraries was performed by sequencing at low depth and analyzing the data using the Bisulfite Sequencing Plugin of the CLC Genomic Workbench. A High mapping rate was obtained with all ccfDNA eluates. B Accurate methylation calls, with 99% bisulfite conversion, were observed.