Protocol

High-Throughput Next Generation Sequencing

Volume 733 of the series Methods in Molecular Biology pp 3-24

Date:

Helicos Single-Molecule Sequencing of Bacterial Genomes

  • Kathleen E. SteinmannAffiliated withHelicos BioSciences Corporation Email author 
  • , Christopher E. Hart
  • , John F. Thompson
  • , Patrice M. Milos

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Abstract

With the advent of high-throughput sequencing technologies, multiple bacterial genomes can be sequenced in days. While the ultimate goal of de novo assembly of bacterial genomes is progressing, changes in the genomic sequence of closely related bacterial strains and isolates are now easily monitored by comparison of their sequences to those of a reference genome. Such studies can be applied to the fields of bacterial evolution, epidemiology, and diagnostics. We present a protocol for single-molecule sequencing of bacterial DNA whose end result is the identification of single nucleotide variants, and various size insertions and deletions relative to a reference genome. The protocol is characterized by the simplicity of sample preparation and the lack of amplification-related sequencing bias.

Key words

Bacterial genome Single-molecule sequencing Sequencing bias SNV PolyA tail HeliScopeTM sequencer