Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey

H.H.D. Kerstens, R.P.M.A. Crooijmans, A. Veenendaal, B.W. Dibbits, T.F.C. Chin-A-Woeng, J.T. den Dunnen, M.A.M. Groenen

Research output: Contribution to journalArticleAcademicpeer-review

68 Citations (Scopus)

Abstract

Background - The development of second generation sequencing methods has enabled large scale DNA variation studies at moderate cost. For the high throughput discovery of single nucleotide polymorphisms (SNPs) in species lacking a sequenced reference genome, we set-up an analysis pipeline based on a short read de novo sequence assembler and a program designed to identify variation within short reads. To illustrate the potential of this technique, we present the results obtained with a randomly sheared, enzymatically generated, 2-3 kbp genome fraction of six pooled Meleagris gallopavo (turkey) individuals. Results - A total of 100 million 36 bp reads were generated, representing approximately 5-6% (~62 Mbp) of the turkey genome, with an estimated sequence depth of 58. Reads consisting of bases called with less than 1% error probability were selected and assembled into contigs. Subsequently, high throughput discovery of nucleotide variation was performed using sequences with more than 90% reliability by using the assembled contigs that were 50 bp or longer as the reference sequence. We identified more than 7,500 SNPs with a high probability of representing true nucleotide variation in turkeys. Increasing the reference genome by adding publicly available turkey BAC-end sequences increased the number of SNPs to over 11,000. A comparison with the sequenced chicken genome indicated that the assembled turkey contigs were distributed uniformly across the turkey genome. Genotyping of a representative sample of 340 SNPs resulted in a SNP conversion rate of 95%. The correlation of the minor allele count (MAC) and observed minor allele frequency (MAF) for the validated SNPs was 0.69. Conclusion - We provide an efficient and cost-effective approach for the identification of thousands of high quality SNPs in species currently lacking a sequenced genome and applied this to turkey. The methodology addresses a random fraction of the genome, resulting in an even distribution of SNPs across the targeted genome
Original languageEnglish
Article number479
JournalBMC Genomics
Volume10
DOIs
Publication statusPublished - 2009

Keywords

  • reduced representation
  • dna-sequences
  • snp discovery
  • association
  • chicken
  • algorithm
  • evolution
  • variants
  • millions
  • database

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