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

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 s...

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Autores principales: Kerstens, Hindrik HD, Crooijmans, Richard PMA, Veenendaal, Albertine, Dibbits, Bert W, Chin-A-Woeng, Thomas FC, den Dunnen, Johan T, Groenen, Martien AM
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772860/
https://www.ncbi.nlm.nih.gov/pubmed/19835600
http://dx.doi.org/10.1186/1471-2164-10-479
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author Kerstens, Hindrik HD
Crooijmans, Richard PMA
Veenendaal, Albertine
Dibbits, Bert W
Chin-A-Woeng, Thomas FC
den Dunnen, Johan T
Groenen, Martien AM
author_facet Kerstens, Hindrik HD
Crooijmans, Richard PMA
Veenendaal, Albertine
Dibbits, Bert W
Chin-A-Woeng, Thomas FC
den Dunnen, Johan T
Groenen, Martien AM
author_sort Kerstens, Hindrik HD
collection PubMed
description 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.
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spelling pubmed-27728602009-11-04 Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey Kerstens, Hindrik HD Crooijmans, Richard PMA Veenendaal, Albertine Dibbits, Bert W Chin-A-Woeng, Thomas FC den Dunnen, Johan T Groenen, Martien AM BMC Genomics Methodology Article 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. BioMed Central 2009-10-16 /pmc/articles/PMC2772860/ /pubmed/19835600 http://dx.doi.org/10.1186/1471-2164-10-479 Text en Copyright © 2009 Kerstens et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Kerstens, Hindrik HD
Crooijmans, Richard PMA
Veenendaal, Albertine
Dibbits, Bert W
Chin-A-Woeng, Thomas FC
den Dunnen, Johan T
Groenen, Martien AM
Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
title Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
title_full Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
title_fullStr Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
title_full_unstemmed Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
title_short Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
title_sort large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772860/
https://www.ncbi.nlm.nih.gov/pubmed/19835600
http://dx.doi.org/10.1186/1471-2164-10-479
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