The gold-standard approach in methylation analysis is the whole genome bisulfite sequencing which gives comprehensive base-pair resolution and quantitative information at most genomic cytosine. This method rely on bisulfite conversion of DNA to detect unmethylated cytosines This method uses cytosine bisulfite conversion converting unmethylated cytosines to uracil followed by random priming, tagging at the 5’ and 3’ end and introduction of Illumina adapters by PCR amplification. Converted bases are identified (after PCR) as thymine in the sequencing data, and read counts are used to determine the % methylated cytosines.
- Discover methylation patterns of all CpG, CHH, and CHG regions across the entire genome.
- Capture full sample diversity with small amounts of DNA
- View methylation at practically every cytosine in the genome across most species
- Submit 5 µg of RNA-free genomic DNA (NGS grade) of molecular weight >40kb, Nanodrop A260/280 ratio >1.8; A260/230=2.0-2.2; and at a concentration of ~50 ng/µl
- Or 5-10 g of fresh tissue fast frozen in liquid nitrogen and shipped in dry ice to eurofins facility.
- RNA free NGS grade gDNA isolation will be carried out using commercially available kit with further purification making it suitable for NGS library preparation.
- Further quality checking will be carried out using agarose electrophoresis and DIN value estimation using Agilent Tape Station. Quantification will be carried out using Qubit 3.0 fluorometer.
- Appropriate amount of gDNA will be bisulfite converted and NGS library will be prepared using TruSeq DNA methylation kit from illumina.
- Library validation will be carried out using High Sensitivity D1000 Screen tape using Agilent Tape station kit.
- Prepared library will be sequenced using 2 x 75 bp using NextSeq 500 or 2 x 125 bp using HiSeq 2500 using latest sequencing chemistries.
- Delivery of data up to 10X-30X coverage of the genome ( depending on size of genome) or customer specified data requirement.
- Sequence data (base call files or bcl files) generated from the sequencer are demultiplexed if required and converted to FASTQ files.
- Raw data will be available for download as a compressed archive of FASTQ files for each sample.
- Raw data can be post processed upon request.
- 5-7 weeks after arrival of your samples and all necessary information. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The TAT is offered expecting no biological or technical difficulties for processing of all project samples