Epigenomics examines the chemical modifications that shape the structure and function of the genome. When epigenomic compounds attach to DNA and modify its function, they are said to have "marked" the genome. These marks do not change the sequence of the DNA. Rather, they change the way cells use the DNA's instructions. The marks are sometimes passed on from cell to cell as cells divide. They also can be passed down from one generation to the next. It regulates gene expression, enabling select genes to be active at different times during the cell life cycle.

Epigenetics plays a significant role in growth, development, and disease progression. The epigenome can be influenced by a variety of factors, including diet, stress, and environmental exposures. These factors can alter epigenetic marks and affect the proteins that write, read, and erase them, potentially leading to changes in gene expression and cellular function.

Eurofins Genomics provides comprehensive wet laboratory and dry laboratory solutions for epigenome and methylome studies, enabling the analysis of genomic responses to various chemical factors through targeted or whole-genome sequencing.

Whole genome bisulfite sequencing (WGBS)

WGBS is considered the gold-standard method for methylation analysis, providing comprehensive, quantitative, and base-pair-resolution data across most genomic cytosines. WGBS is one of the most robust methods for the genome-wide detection of 5-methylcytosine (5mc). This method relies on the bisulfite conversion of DNA to detect unmethylated cytosines. It 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 polymerase chain reaction (PCR) amplification. Converted bases are identified (after PCR) as thymine in the sequencing data, and read counts are used to determine the % methylated cytosines.

Advantage

• Discover methylation patterns of all CpG, CHH, and CHG methylation contexts 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

Methodology

Sample requirement

• Submit 2-4 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.
• Alternatively, 5-10 g of fresh tissue fast frozen in liquid nitrogen and shipped in dry ice to Eurofins Genomics facility.

Sample quality control

• 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 TapeStation.
• Quantification will be carried out using Qubit 3.0 Fluorometer.

Library preparation

• 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 Agilent TapeStation kit.

Sequencing

• Prepared library will be sequenced using 2 x 75 bp using NextSeq500 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.

Deliverables

• Raw data will be available for download as a compressed archive of FASTQ files for each sample.
• Comprehensive compiled report and data set will be shared through secure file-sharing platform.