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Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection.

ABSTRACT: Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 ?g of input DNA per sample.

SUBMITTER: Kenny EM

PROVIDER: S-EPMC3041504 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection.

Kenny Elaine M EM Cormican Paul P Gilks William P WP Gates Amy S AS O'Dushlaine Colm T CT Pinto Carlos C Corvin Aiden P AP Gill Michael M Morris Derek W DW

DNA research : an international journal for rapid publication of reports on genes and genomes 20101216 1

Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal qu ...[more]

PMID: 21163834

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Project description:Macadamia (Macadamia integrifolia, M. tetraphylla and hybrids) is an Australian native nut crop and has a significant economic value in the food industries worldwide. Long juvenility along with traditional breeding strategies impede quick genetic improvement of this crop. The existing cultivars constitute only second to fourth generation of the wild germplasm in the rainforest. The utilisation of molecular markers for genomic selection and genome-wide association studies may accelerate genetic gains. Identification of a robust, reproducible, and cost-effective marker system is instrumental in increasing the efficiency of genomic studies. This study is the first to report the potential of two ultra-high-throughput diversity array technology (DArT) markers (silicoDArT and SNP) in macadamia. Both markers were used to identify the genetic diversity and population structure in 80 macadamia cultivars. Parentage analysis of 25 scions in a rootstock trial was conducted to confirm plant identity where recorded identities did not corroborate with phenotypic field observations. A total of 22,280 silicoDArT and 7,332 SNP markers were reported, of which 11,526 silicoDArT and 3,956 SNP markers were used for analyses after screening with quality control parameters including >95% call rate, >95% reproducibility, and >0.05 one ratio. The average polymorphic information content (PIC) values of silicoDArT and SNP markers were 0.29 and 0.21, respectively. Genetic variance among the cultivars ranged from 0.003 to 0.738 in silicoDArT and 0.004 to 0.412 in SNP markers. Four distinct population groups were identified from SNP data analysis. Most of the accessions used in this study were descended from two or more populations. Cluster analysis clearly separated genotypes of distinct origins, such as the Hawaii Agricultural Experiment Station and Hidden Valley Plantation accessions. Two wild accessions of Macadamia jansenii and M. ternifolia were found to be distantly related to the cultivars. Wild germplasm individuals and their hybrids with cv. '660' formed separate clusters, suggesting that crossing between wild and cultivated genepools can extend genetic diversity. DArTseq-based SNP markers were successfully utilized to confirm the genetic identity of 25 scions in a rootstock trial. Our study suggests that DArT platforms are a robust system for the facilitation of genomic studies with regard to macadamia.

Dataset Information

Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection.

Publications

Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection.

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