title: Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  Six PCR products spread over approximately 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 approximately 142,000 reads, approximately 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.
  
  
  CONCLUSIONS
  Deep 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.
authors.affiliation: Leibniz Institute for Age Research-Fritz Lipmann Institute, Jena, Germany. stau@fli-leibniz.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Taudien
authors.forename: Marco
authors.initials: M
authors.lastname: Groth
authors.forename: Klaus
authors.initials: K
authors.lastname: Huse
authors.forename: Andreas
authors.initials: A
authors.lastname: Petzold
authors.forename: Karol
authors.initials: K
authors.lastname: Szafranski
authors.forename: Jochen
authors.initials: J
authors.lastname: Hampe
authors.forename: Philip
authors.initials: P
authors.lastname: Rosenstiel
authors.forename: Stefan
authors.initials: S
authors.lastname: Schreiber
authors.forename: Matthias
authors.initials: M
authors.lastname: Platzer
mesh_terms: D002898:Chromosomes, Human, Pair 8; D056915:DNA Copy Number Variations; D006239:Haplotypes; D006801:Humans; D005810:Multigene Family; D017422:Sequence Analysis, DNA / Q000379:methods*; D023083:beta-Defensins / Q000235:genetics*
chemical_list: D023083:beta-Defensins
references.citation: BMC Genomics. 2009 Nov 20;10:547
references.pmid: 19930547
references.citation: Nat Genet. 2004 Aug;36(8):861-6
references.pmid: 15247918
references.citation: Hum Mutat. 2008 Oct;29(10):1247-54
references.pmid: 18470942
references.citation: Plant Biotechnol J. 2009 May;7(4):347-54
references.pmid: 19386042
references.citation: PLoS One. 2007 Feb 14;2(2):e197
references.pmid: 17299583
references.citation: Nature. 2005 Sep 15;437(7057):376-80
references.pmid: 16056220
references.citation: BMC Genomics. 2004 Dec 10;5(1):92
references.pmid: 15588320
references.citation: Cytogenet Genome Res. 2008;123(1-4):148-55
references.pmid: 19287149
references.citation: Biotechniques. 2009 Dec;47(6):1023-8
references.pmid: 20041854
references.citation: Nat Genet. 2009 Oct;41(10):1061-7
references.pmid: 19718026
references.citation: BMC Genomics. 2009 Nov 25;10:559
references.pmid: 19939274
references.citation: Biotechniques. 1998 Oct;25(4):684-91
references.pmid: 9793653
references.citation: Am J Hum Genet. 2006 Sep;79(3):439-48
references.pmid: 16909382
references.citation: Genes Cells. 2009 Jan;14(1):69-77
references.pmid: 19077033
references.citation: Am J Gastroenterol. 2010 Feb;105(2):354-9
references.pmid: 19809410
references.citation: Biotechniques. 2009 Mar;46(3):217-23
references.pmid: 19317665
references.citation: Nucleic Acids Res. 2002 Jun 15;30(12):e57
references.pmid: 12060695
references.citation: Nucleic Acids Res. 2007;35(3):e19
references.pmid: 17175532
references.citation: PLoS One. 2009 Jul 20;4(7):e6285
references.pmid: 19617917
references.citation: Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):853-8
references.pmid: 19131514
references.citation: Anal Biochem. 2006 Sep 15;356(2):194-201
references.pmid: 16737678
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1886-91
references.pmid: 19181860
references.citation: Hum Mutat. 2009 Mar;30(3):472-6
references.pmid: 19058222
references.citation: J Negat Results Biomed. 2005 Dec 07;4:9
references.pmid: 16336654
references.citation: Genome Biol. 2009;10(5):R53
references.pmid: 19457243
references.citation: Genome Res. 2008 Nov;18(11):1686-97
references.pmid: 18974263
references.citation: Nucleic Acids Res. 2009 Nov;37(20):6916-26
references.pmid: 19740768
references.citation: Nucleic Acids Res. 1997 Nov 1;25(21):4422-6
references.pmid: 9336479
references.citation: Nucleic Acids Res. 2009 Feb;37(2):e12
references.pmid: 19059995
references.citation: Biotechniques. 2009 Oct;47(4):857-66
references.pmid: 19852769
references.citation: Tumour Biol. 2008;29(2):83-92
references.pmid: 18515986
references.citation: Genomics. 2005 Oct;86(4):423-30
references.pmid: 16039093
references.citation: Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16333-8
references.pmid: 19805302
references.citation: Science. 2006 Nov 17;314(5802):1113-8
references.pmid: 17110569
references.citation: Nat Genet. 2008 Jan;40(1):23-5
references.pmid: 18059266
references.citation: Mucosal Immunol. 2008 Nov;1 Suppl 1:S67-74
references.pmid: 19079235
references.citation: Science. 2007 Oct 19;318(5849):441-4
references.pmid: 17901299
references.citation: Eur J Gastroenterol Hepatol. 2003 Jun;15(6):627-34
references.pmid: 12840673
references.citation: Genome Biol. 2009;10(9):R94
references.pmid: 19747388
references.citation: Am J Hum Genet. 2003 Sep;73(3):591-600
references.pmid: 12916016
references.citation: Nat Med. 2006 Jul;12(7):852-5
references.pmid: 16799556
references.citation: J Immunol Methods. 2006 Jan 20;308(1-2):231-40
references.pmid: 16380128
references.citation: Expert Rev Anti Infect Ther. 2007 Dec;5(6):1049-57
references.pmid: 18039087
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3521-6
references.pmid: 18303113
references.citation: Nature. 2003 Dec 18;426(6968):789-96
references.pmid: 14685227
references.citation: Immunogenetics. 2008 Jan;60(1):1-18
references.pmid: 18193213
references.citation: Nat Rev Immunol. 2003 Sep;3(9):710-20
references.pmid: 12949495