PLoS ONE (Jan 2017)

Single molecule sequencing of the M13 virus genome without amplification.

  • Luyang Zhao,
  • Liwei Deng,
  • Gailing Li,
  • Huan Jin,
  • Jinsen Cai,
  • Huan Shang,
  • Yan Li,
  • Haomin Wu,
  • Weibin Xu,
  • Lidong Zeng,
  • Renli Zhang,
  • Huan Zhao,
  • Ping Wu,
  • Zhiliang Zhou,
  • Jiao Zheng,
  • Pierre Ezanno,
  • Andrew X Yang,
  • Qin Yan,
  • Michael W Deem,
  • Jiankui He

DOI
https://doi.org/10.1371/journal.pone.0188181
Journal volume & issue
Vol. 12, no. 12
p. e0188181

Abstract

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Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.