NGS Applications

Mate-Paired

Samples are run on a 1.2% agarose gel and their concentrations quantified by fluorometry. Four µg of genomic DNA are processed using the Nextera Mate Pair Sample Preparation Kit from Illumina. Specifically, the Gel-Plus pathway is followed. Samples are first tagmented then subjected to strand displacement, purified with Ampure XP beads and subsequently size selected on .6% megabase agarose gel. After the gel cut, samples are purified using the Zymoclean™ Large Fragment DNA Recovery Kit. Samples are then circularized, and linear DNA digested. After shearing the circularized DNA with the Covaris S2 ultrasonicator, samples are bound to Streptavidin beads and end repaired. Samples are then have their 3' ends adenylated and multiplexed adapters ligated. Samples are PCR amplified for 10-15 cycles and purified by Ampure XP beads, and their sizes verified on the Agilent 2100 Bioanalyzer. Samples are quantified by Qubit before being normalized and pooled, then run on the Illumina HiSeq 2500 using Rapid 150PE SBS v3 reagents.

Current Protocol:

Nextera Mate-Pair Sample Preparation Guide