Pick and Choose
Sometimes sequencing the whole genome just gives more information than is really needed. If you want to focus on a particular set of genes or features in the genome, for example, it may be preferable to enrich for these ahead of time, saving the time, effort, and cost of a whole-genome analysis.
MYcroarray offers custom bait libraries designed to selectively capture DNA prior to sequencing. MYbaits target-enrichment kits can contain from hundreds to millions of 80 to 120 mer-long biotinylated RNA bait sequences. These are incubated together in liquid phase with the genomic sample for 36 hours, after which bound sample is pulled out of solution on streptavidin-coated magnetic beads. The baits are then degraded, leaving behind the positively selected DNA that can be used directly for, or amplified prior to, sequencing.
Bait sequences can be designed from genomic libraries or transcriptomes of the same or related species. “Say you’re working on mammals. You can eventually design your baits from the human genome, and then go to fish sequence from more distant genomes—it usually works well down to 90% sequence similarity or so,” said Jean-Marie Rouillard, Ph.D., CSO and co-founder of MYcroarray.
MYbaits is useful for gaining insights into gene structure, even when only transcriptome data is available. From transcriptomic baits the introns flanking the expressed sequences can be pulled down as well. It becomes possible to map exon-intron junctions, as well as to identify noncoding transcriptional and regulatory elements. This, in turn, will allow many fundamental phylogenetic and population questions to be addressed, Dr. Rouillard notes.
MYcroarray presented data in which a set of 14,468 bait sequences designed from western terrestrial garter snake transcriptome sequences were used to enrich genomic DNA prior to PCR amplification and 454 sequencing. 2,556 reference contigs were obtained: in these, 615,338 bases mapped to reference transcripts while 2,161,616 bases mapped to adjacent sequences, meaning that for each base of transcriptome sequence used for baiting an average 3½ bases of new sequence was discovered.
Another group presenting at PAG, led by Aaron Liston, Ph.D., professor at Oregon State University, used MYbaits to enrich for more than 6,000 previously identified polymorphic sites in strawberry, prior to genotyping of 48 F1 offspring. The resulting high-density linkage map allowed them to gain insights into sexual transitions of these and related diploid plant species.