Abstract
Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.
Highlights
Since the launch of the first commercial massively parallel pyrosequencing platform in 2005, next-generation sequencing technology has transformed genomic medicine in both basic and clinical research fronts [1]
On-target efficiency for libraries derived from DNA enriched through RainDance microdroplet-polymerase chain reaction (PCR)
We analyzed on-target efficiency for 3 samples enriched through RainDance microdroplet-PCR
Summary
Since the launch of the first commercial massively parallel pyrosequencing platform in 2005, next-generation sequencing technology has transformed genomic medicine in both basic and clinical research fronts [1]. The past few years have seen wide applications of whole exome sequencing and whole genome sequencing in disease gene discovery, clinical molecular diagnostics and personalized medicine [1]–[7]. Despite the decreasing cost of generation sequencing, whole exome sequencing and whole genome sequencing remain expensive especially when high depth of coverage is needed. Targeted DNA enrichment coupled with generation sequencing allows interrogation of relevant genomic regions at high depth of coverage in a cost-effective manner and is well suited for applications such as molecular diagnosis of diseases with complex but defined genetic etiologies [10]. Microdroplet polymerase chain reaction (PCR) allows simultaneous amplification of up to thousands of target regions through highly multiplexed microfluidic PCR in picoliter reaction volumes [15]
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