Development of a DNA genotyping method using Next-Generation Sequencing and genome-wide DNA polymorphisms analysis

Representative Organization: Tohoku University

2013/08/01 - 2014/03/31

Sendai Bay, coast lines of Japan

Others

A genotyping method using a Next Generation Sequencing platform IonTorrent PGM will be newly developped to evaluate genetic diversity at a genomic scale. Using Pseudopleuronectes yokohamae as a model organisms, the genetic diversity and its dynamics before and after the impact will be investigated based on the spatial and temporal sampling collection.

(1) Whole genome sequencing of Pseudopleuronectes yokohamae The entire genome was sequenced at 33.4*coverage (about 22.41 Gbp). The obtained short sequencing reads of more than 200 M reads were de novo assembled into 525,502 contigs (N50 = 1,994) whiile around 1.1 M reads were not able to be assembled due to its length (too short) and/or repeated regions. (2) SNP typing of the Pseudopleuronectes yokohamae genome by RAD-Seq 1 - 2 M RAD-tag reads were obtained from each individual using RAD-seq and were mapped to the P. yokohamae genome. A total of 440 SNP loci were conserved among the samples studied and successfully genotyped. (3) Development of new microsatellite DNA markers using the Pseudopleuronectes yokohamae genome data Microsatellite sequences were searched in the Pseudopleuronectes yokohamae genome determined earlier, and a total of 330,000 pairs of primers were designed using a program QDD. The screening was performed for 96 pairs of primers ramdomly selected and 18 microsatellite loci were selected according to with the criteria of PCR amplification, polymorphisms and accurate typing. For the 18 microsatellite loci, genotyping was carried out for P. yokohamae individuals collected from Onagawa Bay (40 - 45 individuals). The number of alleles observed was 2 - 25, and the expected and observed heterozygosity was 0.04395 ~ 0.94557 and 0 ~ 0.88889, respectively. This study successfully developed new microsatellite markers, which are applicable for population genetic analyses, using the genome sequencing information of the species of interest. Using the developed microsatellite markers, genotyping will be performed for more than 1500 individuals of P. yokohamae collected from the coastal areas of Japan including Tohoku areas and the genetic diversity and its dynamics will be investigated.

FY2013 Development of a DNA genotyping method using Next-Generation Sequencing and genome-wide DNA polymorphisms analysis