Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations

Sci Rep. 2017 Jun 13;7(1):3356. doi: 10.1038/s41598-017-03448-8.

Abstract

Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / analysis*
  • DNA / genetics
  • DNA Mutational Analysis / methods*
  • Genome, Human
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Mutation*
  • Neoplasm Proteins / genetics*
  • Neoplasms / genetics*
  • RNA / analysis*
  • RNA / genetics
  • RNA Probes / chemistry
  • RNA Probes / genetics

Substances

  • Neoplasm Proteins
  • RNA Probes
  • RNA
  • DNA