Whole genome sequencing

Overview

• Recommended products—curated for whole genome sequencing
• Short workflow—create a normalized library pool in under 4 hours
• Low input, high complexity—up to 3-fold fewer duplicates from 1 ng of DNA when using the xGen DNA Library Prep Kit EZ
• Enzymatic fragmentation—no need for specialized equipment or consumables for DNA fragmentation
• xGen Normalase™ technology—a proprietary enzymatic method of library normalization for multiplexed sequencing
• Automation friendly—works on a variety of platforms

What is whole genome sequencing?

Whole genome sequencing provides comprehensive coverage over the entire mappable genome to gain information about an organism or metagenomic DNA samples. In comparison to targeted sequencing that is often limited to a subset of genes of interest, WGS includes coverage of all coding and non-coding regions, regulatory sequences, and inter- and intra-genic portions of the genome. Then, the sequencing data are aligned to a reference genome for variant analysis, assembled into contigs for de novo genome assemblies, or used for microbial classification (in the case of metagenomic samples).

For low-throughput labs, the 16-reaction product solution for whole genome sequencing is available that includes library prep and adapters. This solution offers a highly streamlined workflow that consists of only three steps:

1. Enzymatic preparation—DNA fragmentation, end-repair, and dA-tailing of dsDNA
2. Adapter ligation
3. PCR amplification

For library prep, the xGen DNA Library Prep Kit EZ uses an enzymatic fragmentation strategy to shear high-quality DNA samples. The kit employs TA ligation-based library prep and includes an enzyme mix for fragmentation, end-repair and dA-tailing, a ligase to attach the xGen Stubby Adapter, as well as a high-fidelity polymerase for indexing PCR. This solution offers premixed xGen UDI Primer Pairs (not included with the xGen DNA Library Prep Kit EZ) for indexing by PCR, which generates an NGS library ready for Illumina® sequencing.

For high-throughput labs, the 96-reaction solution can be used along with the xGen Normalase Module (sold separately), a proprietary enzymatic library normalization kit for multiplexed sequencing. The method doesn't require individual library quantification and utilizes equal volume pooling instead of requiring a different volume for each library when making an equimolar library pool for multiplexed sequencing. The Normalase enzymatic normalization also results in more uniform read depth within a library pool, with a coefficient of variation (CV) <10% when compared to methods that require individual library quantification

Method data

Normalase leads to the formation of balanced library pools without quantification resulting in uniform sequencing data

Normalization using the proprietary Normalase workflow in comparison to the traditional qPCR quantification method provides more uniform results, maximizing the value of the sequencing data as it provides a tighter grouping of reads between samples.

In an experiment where the xGen DNA Library Prep Kit EZ was used in conjunction with Coriell NA12878 genomic DNA (gDNA), xGen Stubby Adapter, xGen UDI primers, and Normalase, the CV was lower (6.9%) compared to when libraries were quantified by qPCR (18%, Figure 1). This result indicates that the use of Normalase provides a faster, more efficient workflow for multiplex sequencing.

In a shotgun metagenomic sequencing experiment, libraries were constructed using the xGen DNA Library EZ Kit and 1 ng of DNA derived from a mock metagenome DNA standard (ATCC, MSA-1000™) with varying GC content (30⁠–⁠68.8%). The kit provided comprehensive normalized coverage across distinct GC compositions and coverage uniformity (Figure 2A). The data also show that the libraries pooled using the Normalase workflow maintain a high⁠-⁠quality sequencing coverage across a wide range of GC targets (Figure 2B).

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