Whole Exome Sequencing

Exome sequencing is a powerful approach for discovery research in human health, agriculture, and environmental science.  For example, by specifically targeting and sequencing the 1.5% of the human genome comprised of protein-coding and other interesting regions, researchers are able to quickly access 85% of the mutations that play critical roles in disease. Exome sequencing leverages the power of next-generation sequencing technology both in terms of cost-effectiveness and turn around time.

Challenges

Choosing the appropriate enrichment method and sequencing depth is critical to carrying out a successful project. The Centrillion team works with you to ensure your exome sequencing projects are completed on time and cost-effectively. We work closely with you to understand your project goals and provide suggestions for optimizing enrichment and coverage to stay within your budget. We keep you informed as your project progresses through the laboratory workflow.

Workflow

lab6

Sample QC

Input nucleic acid requirements
– Minimum 1ug at 100ng/ul
– FFPE samples, minimum 2 ug at <135ul total volume

QC methods
– Qubit
– Picogreen Method

Library Preparation

SureSelect® Human Exon V6 (Agilent)

Qualified gDNA
DNA Fragmentation
End Repair
A Tailing
Adapter Ligation
Size Selection – Beads
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 not recommended for this application
Coverage depth
– 30x, 50x, 100x, 200x, Custom
Read length
– 100bp paired ends, Custom
Sequencing quality
– >80% of bases above Q30

Analysis and Delivery

Analysis
– Align reads to reference genome
– Identify variants, SNVs, SNPs, short in/dels
– Compare to the latest dbSNP
– Annotate position to gene i.e., intron, exon, UTR
– Annotate SNP class i.e., synonymous vs. non-synonymous
– Annotate to important genomic features, i.e., GWAS SNPs, conserved genomic elements
– Create FastQ files, BI summary report
Delivery
– FTP or external data drive
– Data storage < 30 days after initial data delivery

To discuss your project contact us.