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Nucleic Acids Research Advance Access published online on November 15, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm918
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© 2007
The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

Gagan A. Pandya1, Michael H. Holmes1, Sirisha Sunkara1, Andrew Sparks2, Yun Bai1, Kathleen Verratti1, Kelly Saeed1, Pratap Venepally1, Behnam Jarrahi1, Robert D. Fleischmann1 and Scott N. Peterson1,*

1Pathogen Functional Genomics Resource Center, The Institute for Genomic Research at the J. Craig Venter Institute, Rockville, MD 20850, USA and 2Affymetrix, Inc., Santa Clara, CA 95051, USA

*To whom correspondence should be addressed. Tel: +1 301 795 7539; Fax: +1 301 838 0208; Email: scottp{at}jcvi.org

Received August 13, 2007. Revised September 21, 2007. Accepted October 9, 2007.

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.

Present address: Andrew Sparks, Complete Genomics, 658 North Pastoria Avenue, Sunnyvale, CA 94085, USA.


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