An article in BMC Plant Methods presents 3D-GBS: an optimized genotyping-by-sequencing procedure, to provide an ultra-high-throughput and ultra-low-cost genotyping solution for species with small to medium-sized genome.
], using a rare cutter with either 33% or 66% of GC will inevitably induce variable density of restriction fragments across chromosomes and species. On the other hand, frequent cutters can have a 50% GC content, such as MspI or BfaI , allowing a more even distribution of restriction fragments throughout the genome, as illustrated by Torkamaneh et al. [].
In light of the above, we explored the idea of improving the two-enzyme approach by using a second rare cutter, such as NsiI [], with a cutting site differing in GC content and exploiting methylation insensitivity to capture hypermethylated regions missed by PstI. The combination of NsiI with PstI and MspI presented a good opportunity to obtain a sufficient and efficient low density of SNPs distributed more evenly in the genome.
]. For average minor allele frequency and nucleotide diversity values, equivalent results were provided across the entire range of reads per sample, suggesting that even with a very limited sequencing effort one can perform high-quality genetic diversity analysis.Full size table The distribution of the SNPs on the genetic map was very similar from 100 to 300K reads while, with only 50K reads per sample, large gaps were detected and some chromosome extremities were missed . While the density of markers doubled between 100 and 300K reads, the distribution of the SNPs across the genetic map remained very similar with some regions that were denser in SNPs using 300K reads .
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