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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...
Autores principales: | , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2009
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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. |
format | Text |
id | pubmed-2772860 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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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