Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing

BMC Genomics

Research Article

Jochen Hampe¹  Philip Rosenstiel²  Stefan Schreiber³  Andreas Petzold⁴  Marco Groth⁴  Matthias Platzer⁴  Stefan Taudien⁴  Klaus Huse⁴  Karol Szafranski⁴ 

[1] Dept. of General Intermal Medicine, Christian-Albrechts-University, D-24105, Kiel, Germany;Institute of Clinical Molecular Biology, Christian-Albrechts-University, D-24105, Kiel, Germany;Institute of Clinical Molecular Biology, Christian-Albrechts-University, D-24105, Kiel, Germany;Dept. of General Intermal Medicine, Christian-Albrechts-University, D-24105, Kiel, Germany;Leibniz Institute for Age Research - Fritz Lipmann Institute, D-07745, Jena, Germany;

关键词: Next Generation Sequencing;    Single Nucleotide Polymorphism;    Amplicon Sequencing;    Diploid Genome;    Copy Number Estimation;   

DOI  :  10.1186/1471-2164-11-252

 received in 2010-01-22, accepted in 2010-04-19,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundThe beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel.ResultsSix PCR products spread over ~87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From ~142,000 reads, ~120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed.ConclusionDeep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.

【 授权许可】

CC BY   
© Taudien et al; licensee BioMed Central Ltd. 2010