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sequencing intractable dna to close microbial genomes测序的微生物基因组dna关闭
Sequencing Intractable DNA to Close Microbial Genomes
Richard A. Hurt Jr., Steven D. Brown, Mircea Podar, Anthony V. Palumbo, Dwayne A. Elias*
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America
Abstract
Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome
sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in
microbial genomes are ruled ‘‘intractable’’ resulting in a growing number of genomes with sequence gaps deposited in
databases. A procedure was developed to sequence such problematic regions in the ‘‘non-contiguous finished’’
Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap).
The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to
amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle
supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene
annotation, and provide a step-wise method that reduces the effort required for genome finishing.
Citation: Hurt RA Jr., Brown SD, Podar M, Palumbo AV, Elias DA (2012) Sequencing Intractable DNA to Close Microbial Genomes. PLoS ONE 7(7): e41295.
doi:10.1371/journal.pone.0041295
Editor: Sarah K. Highlander, Baylor College of Medicine, United States of America
Received April 9, 2012; Accepted June 19, 2012; Published July 31, 2012
Copyright: 2012 Hurt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distr
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