Targeted sequencing library preparation by genomic DNA circularization

Samuel Myllykangas,Georges Natsoulis,Hanlee P Ji,John M Bell

doi:10.1186/1472-6750-11-122

Abstract

BackgroundFor next generation DNA sequencing, we have developed a rapid and simple approach for preparing DNA libraries of targeted DNA content. Current protocols for preparing DNA for next-generation targeted sequencing are labor-intensive, require large amounts of starting material, and are prone to artifacts that result from necessary PCR amplification of sequencing libraries. Typically, sample preparation for targeted NGS is a two-step process where (1) the desired regions are selectively captured and (2) the ends of the DNA molecules are modified to render them compatible with any given NGS sequencing platform.ResultsIn this proof-of-concept study, we present an integrated approach that combines these two separate steps into one. Our method involves circularization of a specific genomic DNA molecule that directly incorporates the necessary components for conducting sequencing in a single assay and requires only one PCR amplification step. We also show that specific regions of the genome can be targeted and sequenced without any PCR amplification.ConclusionWe anticipate that these rapid targeted libraries will be useful for validation of variants and may have diagnostic application.

Highlights

For generation DNA sequencing, we have developed a rapid and simple approach for preparing DNA libraries of targeted DNA content
We have developed an approach in which circularization of a specific genomic DNA molecule directly incorporates Illumina sequencing library components (Figure 1)
Targeted genomic circularization requires a restriction endonuclease digest of nanogram amounts of genomic DNA followed by incubation with a mixture of two types of oligonucleotides

Summary

Introduction

For generation DNA sequencing, we have developed a rapid and simple approach for preparing DNA libraries of targeted DNA content. Targeted resequencing has proven useful for a large number of applications including validating variants from whole genome sequencing, studying disease-relevant gene subsets and diagnostic detection of clinically actionable variants. Along these lines, a variety of methods have been developed to enrich specific regions of the genome. Oligonucleotide-Selective Sequencing which is a targetspecific hybridization and extension approach for capturing genomic target sequence has been developed [14]. Most of these methods require the added step of ligating sequencing adapters either pre- or postenrichment

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