Reduced Representation Bisulphite Sequencing (RRBS)

Reduced Representation Bisulphite Sequencing (RRBS) is a practical and cost-effective method designed to efficiently yield genome-wide quantitative DNA methylation information at single base resolution. Enriching for areas of the genome with high CpG content reduces the number of sequencing reads required which significantly reduces the cost of methylome analysis compared to whole genome bisulphite sequencing approaches.

Challenges

Low base diversity resulting from restriction digestion and enrichment is a major challenge in obtaining excellent targeted bisulfite sequencing results. The Ovation(R) RRBS Methyl-Seq System addresses this problem by incorporating a variable number of adaptor-derived random bases at the beginning of the read to introduce sufficient diversity to ensure efficient clustering and run initiation aswell as supporting de-phasing the sequence data.

Centrillion will help you determine the required coverage, identify the number of samples needed, and advise on the number of samples that can be multiplexed per lane. Laboratory analytics are carried out throughout the process to ensure that high quality data is produced.

Workflow

lab3

Sample QC

DNA input requirements
– Minimum of 100ng at 12ng/ul, min volume of 10ul

QC methods
– Qubit® dsDNA BR

RNA Library Prep

Ovation® RRBS Methy-Seq System (NuGEN)

Quaified gDNA
Mspl digestion
Adaptor Ligation
Repair
Bisulphite conversion
PCR Amplification
QC Library

Library Quality and Quantity Control

Library QC methods
– BioAnalyzer® (Agilent): Fragment size
– qPCR (Kapa®): Concentration, equimolar pooling
Or
– ddPCR (Biorad): Fragment size and quantity

Library QC pass criteria
– Min of 4nM
– Fragment size range

Sequencing

Available platforms
– HiSeq 2500
– MiSeq
Coverage depth in millions of reads
– 10x, custom
Read length
– 100bp paired ends, custom
Sequencing quality
– >80% of bases above Q30

Analysis and Delivery

Analysis
– Raw reads preprocessing and quality control
– Read alignment with human reference genome, GRCh37 – http://grch37.ensembl.org
– Extraction of methylation regions and levels
– Differential methylation analysis
Delivery
– FTP or external data drive
– Data storage < 30 days after initial data delivery

To discuss your project contact us.