NGS Applications

Exome Capture

Samples are run on a 1.2% agarose gel and their concentrations quantified by fluorometry. Following the SureSelectXT Human All Exon V4 Kit protocol, 3 µg genomic DNA is sonicated using the Covaris S2 ultrasonicator then purified and assessed on the Agilent 2100 Bioanalyzer. Samples are end repaired, 3' ends adenylated and then barcoded with truncated adapters. Samples are PCR amplified then purified with Ampure XP beads and validated on the Agilent 2100 Bioanalyzer. 750 ng of the fragment library is vacuumed to 3.5 µl then hybridized and captured with the SureSelect Hybridization reagents. During post-hybridization amplification, index tags are added to the adapters. Samples are again purified with Ampure XP beads, then checked on the Agilent 2100 Bioanalyzer to ensure the library is the expected size. Samples are quantified by Qubit before being normalized and pooled, then run on the Illumina HiSeq 2500 using PE100 SBS v3 reagents.

 

Current Protocol:

SureSelect XT v4 Sample Preparation Guide

Past Protocols:

Instructions for Performing the Nextera Rapid Capture Protocol

TruSeq Sample Enrichment Guide

 

Exome Metrics

Total PE ReadsMean CoveragePCT Target Bases >=10XPCT Target Bases >=20X
50,000,000 70 98% 93%
60,000,000 80 99% 95%
80,000,000 100 99% 97%
100,000,000 125 99%  98%