Format

Send to

Choose Destination
See comment in PubMed Commons below
PLoS One. 2012;7(7):e41295. doi: 10.1371/journal.pone.0041295. Epub 2012 Jul 31.

Sequencing intractable DNA to close microbial genomes.

Author information

1
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.

PMID:
22859974
PMCID:
PMC3409199
DOI:
10.1371/journal.pone.0041295
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Public Library of Science Icon for PubMed Central
    Loading ...
    Support Center