Plant & Animal Genomes XVII Conference

January 10-14, 2009
Town & Country Convention Center
San Diego, CA

Isolation Of Hundreds Of Selected Gene Segments In Hundreds Of Diverse DNA Samples For Pooled 454 Sequencing

Andy Flavell¹ , Naeem Syed¹ , Linda Cardle² , Micha Bayer² , David Marshall² , Robbie Waugh²

¹  Division of Plant Sciences, University of Dundee at SCRI, Invergowrie, Dundee DD2 5DA, Scotland, UK

²  Genetics Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

Studies of genetic diversity in plants and animals use either molecular markers or DNA sequencing. High throughput SNP genotyping offers great accuracy and detail that has been successful for population and association genetic studies in humans and some other mammals where tens of thousands of SNPs are available. Unfortunately, most animals and plants do not have this luxury in genomic resource available to the scientist and SNP sets become less effective in describing the higher diversity found in wild or semi-domesticated populations. An alternative approach is to replace marker analysis by next generation sequencing (NGS), thus effectively ‘genotyping by sequencing’. NGS samples huge numbers of pooled DNAs in parallel. But what if you want to sequence lots of samples at lots of loci? In many crop systems and in wide diversity studies even sub-pools of a 454 plate are too costly, so samples need to be pooled, necessitating the use of sequence tags to define the genome of origin. However, tags means separate PCRs per sample - if you want to sequence, say, 200 loci in 100 samples (that’s very roughly a complete 400,000 454 run) that means 20,000 PCRs and at least 4000 tagged primers, costing at least $15,000 even before you start thinking about the sequencing cost. We describe a PCR-based method that can achieve the same result using 500 PCRs and 500 primers, saving LOTS of time and money.