Limits...
Detection of genomic variation by selection of a 9 mb DNA region and high throughput sequencing.

Nikolaev SI, Iseli C, Sharp AJ, Robyr D, Rougemont J, Gehrig C, Farinelli L, Antonarakis SE - PLoS ONE (2009)

Bottom Line: Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold.We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA.Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland. Sergey.Nikolaev@unige.ch

ABSTRACT
Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb) and 7 (1.1 Mb) from an individual from the International HapMap Project (NA12872). We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage > or = 4-fold, and 97.9% concordant in regions with coverage > or = 15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

Show MeSH

Related in: MedlinePlus

Proportion of different genomic elements in the captured samples with and without COT-I DNA as compared to their relative abundance in the human genome.Reads were mapped to the reference human genome using BLAT, allowing 2 mismatches and 1 indel with a minimal perfect match of 30 bp.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2722027&req=5

pone-0006659-g002: Proportion of different genomic elements in the captured samples with and without COT-I DNA as compared to their relative abundance in the human genome.Reads were mapped to the reference human genome using BLAT, allowing 2 mismatches and 1 indel with a minimal perfect match of 30 bp.

Mentions: Next we compared the results of hybridizations with and without COT-1 DNA. In order to better understand the impact of COT-1 DNA on the enrichment we assessed how efficiently it competes for hybridization with repetitive elements. We compared the proportion of Illumina reads that map to different kinds repeats (with and without COT) to the proportion of these elements in a genome (Fig. 2). Generally, addition of COT-1 DNA during array hybridization reduced the proportion of repetitive elements present in the captured DNA by 33%, thereby increasing the percentage of reads from non-repetitive genomic elements, and improving enrichment for the target sequence ∼14-fold.


Detection of genomic variation by selection of a 9 mb DNA region and high throughput sequencing.

Nikolaev SI, Iseli C, Sharp AJ, Robyr D, Rougemont J, Gehrig C, Farinelli L, Antonarakis SE - PLoS ONE (2009)

Proportion of different genomic elements in the captured samples with and without COT-I DNA as compared to their relative abundance in the human genome.Reads were mapped to the reference human genome using BLAT, allowing 2 mismatches and 1 indel with a minimal perfect match of 30 bp.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2722027&req=5

pone-0006659-g002: Proportion of different genomic elements in the captured samples with and without COT-I DNA as compared to their relative abundance in the human genome.Reads were mapped to the reference human genome using BLAT, allowing 2 mismatches and 1 indel with a minimal perfect match of 30 bp.
Mentions: Next we compared the results of hybridizations with and without COT-1 DNA. In order to better understand the impact of COT-1 DNA on the enrichment we assessed how efficiently it competes for hybridization with repetitive elements. We compared the proportion of Illumina reads that map to different kinds repeats (with and without COT) to the proportion of these elements in a genome (Fig. 2). Generally, addition of COT-1 DNA during array hybridization reduced the proportion of repetitive elements present in the captured DNA by 33%, thereby increasing the percentage of reads from non-repetitive genomic elements, and improving enrichment for the target sequence ∼14-fold.

Bottom Line: Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold.We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA.Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland. Sergey.Nikolaev@unige.ch

ABSTRACT
Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb) and 7 (1.1 Mb) from an individual from the International HapMap Project (NA12872). We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage > or = 4-fold, and 97.9% concordant in regions with coverage > or = 15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

Show MeSH
Related in: MedlinePlus