National Institute Of Biomedical Genomics (NIBMG)

National Institute Of Biomedical Genomics (NIBMG) National Institute Of Biomedical Genomics National Institute Of Biomedical Genomics

Present Status

Exome Capture and Deep Resequencing

Exome capture and deep resequencing are being performed for genomic DNA from tumor and whole blood samples from 500 patients (N = 1000) using NimbleGen 2.1M Exome Array (NimbleGen, Roche) and GS FLX Titanium sequencing (454, Roche). The basic steps include:

Preparation of single stranded DNA library by fragmentation of genomic DNA and ligation of adapters to fragment ends.
Exome capture by hybridization of exome fragments to oligonucleotide probes located in microarray or in solution, removal of non exomic fragments and elution of captured exome fragments.
Emulsion-PCR of enriched single stranded DNA library on DNA binding beads in oil-water emulsion based micro reactors followed by recovery of beads bearing clonally amplified library.
Flow-cell sequencing of the clonally amplified library in pico titer plates in GS-FLX 454 DNA sequencers.

Exome Capture

Genomic DNA samples (pairs of tumor and whole blood DNA from patients) from ACTREC are being processed.
Genomic DNA of cell lines COLO-829 and COLO-829 BL from Wellcome Trust-Sanger Institute are being used for calibration of sequencing analyses programs and QA/QC of analyzed data in NIBMG.
SOP including QA/QC criteria for acceptability of exome capture results has been established.
The extent of exome enrichment of captured DNA samples are being evaluated by SYBR-Green real time PCR based relative quantitation in 4 different genes.

Flow-Cell Sequencing

SOP including QA/QC criteria for acceptability of sequencing data as well as sequencing analysis has been established and implemented.
A completely automated pipeline, from image file input after completion of a sequence run to QA/QC of sequence data output, has been developed and put to routine use.
The median read length obtained till date is 363 quality passed bases and on an average 491 million bases of quality passed sequence data are being obtained per run (403 MB to 610 MB).

Sequence coverage of targeted exome at varying levels of sequence data size.

Sequence coverage of target at varying depth of coverage.

Number of high confidence single nucleotide variations (SNVs) detected at varying depth of coverage.

Genome Wide Scan

Genome wide scan of the samples are being performed with Illumina Omni-Quad bead chips which provides genotype information on 1.14 million SNP loci per sample. The protocol for sample processing has been optimized and 4 genomic DNA samples from patients as well as the two cell line DNA samples in duplicate (for QA/QC) has been processed. The SOP including QA/QC parameters is being currently prepared. For these samples, call rates ranged from 0.955 to 0.995. Average inter chip concordance rate of 99.05% was observed in the generated data. Further analysis of the genotype data is in progress.

ICGC-India Project Team @ NIBMG:

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