title: Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.
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.
authors: Division of Genomic Diagnostics and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
authors: Zhenming
authors: Z
authors: Yu
authors: Division of Genomic Diagnostics and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
authors: Kajia
authors: K
authors: Cao
authors: Division of Genomic Diagnostics and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
authors: Tanya
authors: T
authors: Tischler
authors: Division of Genomic Diagnostics and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America.
authors: Catherine A
authors: CA
authors: Stolle
authors: Division of Genomic Diagnostics and Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors: Avni B
authors: AB
authors: Santani
mesh_terms: D004277:DNA, Single-Stranded / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D044085:Microfluidics / Q000379:methods*; D010940:Plant Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; C526101:Mung Bean Nuclease; D010940:Plant Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases