Abstract
Next generation DNA sequencing (NGS) technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap) method generate datasets that are comparable to standard amount (SA) whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes.
Highlights
The advent of massively parallel sequencing technologies has enabled the rapid and cost effective resequencing of cancer genomes and exomes [1,2,3]
By benchmarking the performance of sequencing libraries generated using this procedure, Minimal Starting Amount Capture Method (MSA-Cap), against widely accepted criteria we find that our exome capture method generates high quality data that is comparable with sequencing libraries generated using microgram amounts of starting material
A modified exome capture method To facilitate targeted resequencing from minimal amounts of starting DNA, we designed a method, Minimal Starting Amount Capture (MSA-Cap), that is able to utilize as little as 50 ng starting DNA (Figure 1, Methods S1)
Summary
The advent of massively parallel sequencing technologies has enabled the rapid and cost effective resequencing of cancer genomes and exomes [1,2,3]. Despite the constant improvement of NGS technologies, one limitation is the amount of starting material that is often required. Standard protocols for targeted resequencing of the human exome using sequence capture [4] require, on average, one to three mg of starting DNA. These sequence capture methods involve DNA fragmentation, end repair and A-tailing of the fragmented DNA followed by hybridization to RNA (in case of the Agilent SureSelect system) or DNA baits (in case of the NimbleGen or Illumina systems) that are designed to physically capture specific DNA sequences. With the increasing use of small core biopsies [5] and the requirement to perform multiple types of analysis on a single sample (e.g. genetic, genomic, transcriptomic, proteomic and histological analysis), the limitations of DNA recovery from biopsies are exacerbated
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