Whole Genome Sequencing

High-throughput next generation sequencing technologies have made whole genome sequencing (WGS) accessible and affordable. WGS generates an enormous amount of data and when placed in the context of a comparative study it can be the gateway to discovering clinically relevant genomic associations between genetic variations and phenotypes. WGS in de novo sequencing projects can reveal surprising similarities and differences between organisms that have been grouped or classified based on morphological and functional observations. While challenging, de novo studies can reveal exciting new discoveries.

Challenges

Good quality, intact genomic DNA in sufficient quantities is required for obtaining high quality sequencing results. Post-sequencing data analysis can be daunting, especially for de novo sequencing projects (no reference genomic data available). At Centrillion we have many years of experience designing WGS experiments, preparing WGS libraries and sequencing. Typically, for organisms without reference databases we provide alignment and deliver Fastq files. If required, we can assist with detailed, customized bioinformatics support tailored to your research needs. Using standard analysis software, when relevant reference databases are available we can address specific questions about SNVs, indels etc. We offer WGS at low depth (2-5X) or deeper coverage (30X to 100X+) for human, mouse, microbial, viral, and non-model organisms.

Workflow

lab

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

DNA Fragmentation
End Repair
A Tailing
Adapter Ligation
Size Selection – Beads
QC Library

Library Quality and Quality Control

Library QC methods
– BioAnalyzer: Fragment size
– Kapa qPCR: Concentration, equimolar pooling

– ddPCR: Fragment size and quantity

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

Sequencing

Available platforms
– HiSeq 2500
– MiSeq
Coverage depth
– 2-5x, 30x-100x, Custom
Read length
– 100bp paired ends, Custom
Sequencing quality
– >80% of bases above Q30

Data Analysis

Analysis
– Align reads to reference genome
– De novo assembly, custom bioinformatics,   inquire
– Create FastQ files and summary report
Delivery
– FTP or external data drive
– Data storage < 30 days after initial data delivery

To discuss your project contact us.