Dataset Information

Genome sequencing in microfabricated high-density picolitre reactors.

ABSTRACT: The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

SUBMITTER: Margulies M

PROVIDER: S-EPMC1464427 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Genome sequencing in microfabricated high-density picolitre reactors.

Margulies Marcel M Egholm Michael M Altman William E WE Attiya Said S Bader Joel S JS Bemben Lisa A LA Berka Jan J Braverman Michael S MS Chen Yi-Ju YJ Chen Zhoutao Z Dewell Scott B SB Du Lei L Fierro Joseph M JM Gomes Xavier V XV Godwin Brian C BC He Wen W Helgesen Scott S Ho Chun Heen CH Irzyk Gerard P GP Jando Szilveszter C SC Alenquer Maria L I ML Jarvie Thomas P TP Jirage Kshama B KB Kim Jong-Bum JB Knight James R JR Lanza Janna R JR Leamon John H JH Lefkowitz Steven M SM Lei Ming M Li Jing J Lohman Kenton L KL Lu Hong H Makhijani Vinod B VB McDade Keith E KE McKenna Michael P MP Myers Eugene W EW Nickerson Elizabeth E Nobile John R JR Plant Ramona R Puc Bernard P BP Ronan Michael T MT Roth George T GT Sarkis Gary J GJ Simons Jan Fredrik JF Simpson John W JW Srinivasan Maithreyan M Tartaro Karrie R KR Tomasz Alexander A Vogt Kari A KA Volkmer Greg A GA Wang Shally H SH Wang Yong Y Weiner Michael P MP Yu Pengguang P Begley Richard F RF Rothberg Jonathan M JM

Nature 20050731 7057

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately ...[more]

PMID: 16056220

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Project description:Few public sector rice breeders have the capacity to use NGS-derived markers in their breeding programmes despite rapidly expanding repositories of rice genome sequence data. They rely on > 18,000 mapped microsatellites (SSRs) for marker-assisted selection (MAS) using gel analysis. Lack of knowledge about target SNP and InDel variant loci has hampered the uptake by many breeders of Kompetitive allele-specific PCR (KASP), a proprietary technology of LGC genomics that can distinguish alleles at variant loci. KASP is a cost-effective single-step genotyping technology, cheaper than SSRs and more flexible than genotyping by sequencing (GBS) or array-based genotyping when used in selection programmes. Before this study, there were 2015 rice KASP marker loci in the public domain, mainly identified by array-based screening, leaving large proportions of the rice genome with no KASP coverage. Here we have addressed the urgent need for a wide choice of appropriate rice KASP assays and demonstrated that NGS can detect many more KASP to give full genome coverage. Through re-sequencing of nine indica rice breeding lines or released varieties, this study has identified 2.5 million variant sites. Stringent filtering of variants generated 1.3 million potential KASP assay designs, including 92,500 potential functional markers. This strategy delivers a 650-fold increase in potential selectable KASP markers at a density of 3.1 per 1 kb in the indica crosses analysed and 377,178 polymorphic KASP design sites on average per cross. This knowledge is available to breeders and has been utilised to improve the efficiency of public sector breeding in Nepal, enabling identification of polymorphic KASP at any region or quantitative trait loci in relevant crosses. Validation of 39 new KASP was carried out by genotyping progeny from a range of crosses to show that they detected segregating alleles. The new KASP have replaced SSRs to aid trait selection during marker-assisted backcrossing in these crosses, where target traits include rice blast and BLB resistance loci. Furthermore, we provide the software for plant breeders to generate KASP designs from their own datasets.

Dataset Information

Genome sequencing in microfabricated high-density picolitre reactors.

Publications

Genome sequencing in microfabricated high-density picolitre reactors.

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