Genotyping by sequencing (GBS)

Genotyping by sequencing comes in different flavors. Most of the methods are based on the availability of a genome sequence of the species under consideration. The genome can be in a primitive draft stage, composed of unassembled contigs/scaffolds.

• A first approach is based on constructing a sequencing library with reduced representativety from the genome of interest. With other words, only a part of the genome is sequenced in order to reduce sequencing cost. The part of the genome sequenced is a random selection of genomic regions, based on restriction sites, but the random selection is consistent between samples. Sequencing depth has to be high enough to have a high chance to observe both alleles for heterozygous sites.
• A second approach is based on high multiplexing of targeted amplicons. The sequencing cost is depending on the number of targeted variants. As a pool of probes primer has to be designed and synthetized this approach is only suited for relatively high number of samples to be cost effective.
• A third approach is based on so called shallow sequencing. In contrast to WGS (whole genome sequencing) at high coverage, e.g.30X, where the average number of times any locus in the genome is sequence is 30 times, and genotypes can be called with high accuracy, shallow sequencing is typically done at 1X or even lower. Prior knowledge of variants in the species is an advantage but not a necessity. If enough individuals are sequenced at low coverage, the pooled sequences across individuals can be used in variant discovery step. As a result of the shallow sequencing, it will not be possible to call genotypes with certainty but instead, genotype probabilities will be generated that are less informative than genotypes. The loss of informatively on individual sites will be more than compensated by the number of variant that are interrogated.
• The last and ultimate approach is WGS (whole genome sequencing) at high coverage e.g.30X. Clearly the most comprehensive method, WGS is still expensive, but cost continue to drop fast.