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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC RESEARCH TO ENHANCE EFFICIENT AND SUSTAINABLE PRODUCTION OF BEEF CATTLE AND SHEEP

Location: Genetics, Breeding, & Animal Health

Title: Low-cost genotyping by next generation sequencing: Next generation genotyping

Authors
item Curry, John -
item Snelling, Warren
item Lindholm-Perry, Amanda
item Dier, Paul -
item Shin, Maria -
item Nguyen, Jessica -
item Bulsara, Nadeem -
item Thallman, Richard
item Fofanov, Viacheslav -
item Koshinsky, Heather -

Research conducted cooperatively with:
item

Submitted to: Plant and Animal Genome Conference
Publication Type: Abstract Only
Publication Acceptance Date: November 9, 2012
Publication Date: January 12, 2013
Citation: Curry, J.D., Snelling, W.M., Lindholm-Perry, A.K., Dier, P., Shin, M., Nguyen, J., Bulsara, N., Thallman, R.M., Fofanov, V.Y., Koshinsky, H. 2013. Low-cost genotyping by next generation sequencing: Next generation genotyping [abstract]. Plant and Animal Genome XXI Conference. Poster Abstract No. P0525.

Technical Abstract: The utility of mass genotyping has been tempered by both the expense and time requirements of existing technologies, particularly for larger scale projects. We have demonstrated the ability to use next generation sequencing technologies to genotype thousands of DNA samples for hundreds of loci – a next generation genotyping technology (NGG). The power and economy of NGG is that it focuses sequence read generation on the loci to be genotyped. NGG is a ligation-dependent single well PCR that uses allele barcodes contained within the ligation probes as well as sample barcodes added by PCR. A library suitable for short next generation sequence data generation on a single GAIIx lane on hundreds of loci in thousands of samples can be created in 24 hours. The barcoded sequence on the reads are binned and counted. The bins provide the sample, locus, and allele identification. The read counts are used to infer the genotype. The cost per sample is highly competitive. The sample barcodes allow different assays to be mixed for sequence data generation. The most cumbersome step is DNA extraction. The NGG assay has been used to interrogate simple and poly allelic SNPs, INDELs, and STR. In NGG experiments on cattle parentage with 95 loci on 1080 B. taurus samples, we have obtained 99.1% concordance and 96.1% call rate with genotype data previously identified using a panel of 50,000 SNP.

Last Modified: 10/20/2014
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