Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey
<p>Abstract</p> <p>Background</p> <p>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 refere...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2009-10-01
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| Series: | BMC Genomics |
| Online Access: | http://www.biomedcentral.com/1471-2164/10/479 |
| _version_ | 1818111705317638144 |
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| author | den Dunnen Johan T Chin-A-Woeng Thomas FC Dibbits Bert W Veenendaal Albertine Crooijmans Richard PMA Kerstens Hindrik HD Groenen Martien AM |
| author_facet | den Dunnen Johan T Chin-A-Woeng Thomas FC Dibbits Bert W Veenendaal Albertine Crooijmans Richard PMA Kerstens Hindrik HD Groenen Martien AM |
| author_sort | den Dunnen Johan T |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 <it>Meleagris gallopavo </it>(turkey) individuals.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p> |
| first_indexed | 2024-12-11T03:07:17Z |
| format | Article |
| id | doaj.art-a232bf8900454b42b1c85788fcd0e99a |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-11T03:07:17Z |
| publishDate | 2009-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-a232bf8900454b42b1c85788fcd0e99a2022-12-22T01:22:56ZengBMCBMC Genomics1471-21642009-10-0110147910.1186/1471-2164-10-479Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkeyden Dunnen Johan TChin-A-Woeng Thomas FCDibbits Bert WVeenendaal AlbertineCrooijmans Richard PMAKerstens Hindrik HDGroenen Martien AM<p>Abstract</p> <p>Background</p> <p>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 <it>Meleagris gallopavo </it>(turkey) individuals.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p>http://www.biomedcentral.com/1471-2164/10/479 |
| spellingShingle | den Dunnen Johan T Chin-A-Woeng Thomas FC Dibbits Bert W Veenendaal Albertine Crooijmans Richard PMA Kerstens Hindrik HD Groenen Martien AM Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey BMC Genomics |
| 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 |
| url | http://www.biomedcentral.com/1471-2164/10/479 |
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