| Design Description | The current Japanese crested ibis population in Japan is originated from the only five founder individuals (2 males and 3 females) donated by China. For the discovery of genome wide SNP and STR markers in Japanese crested ibis, we generated sequences from the Japanese crested ibis RRLs using the next generation sequencer Hiseq2000 (Illumina). Also, we simultaneously obtained the genotype data on each of markers by sequencing the 5 RRLs independently prepared from the 5 founder genomes. At the time of this study, the HiSeq2000 produced sequences of 150-200 million DNA fragments with 100bp read length in one sequencing lane. That allowed us to analyze DNA fragments from 0.5-1 million loci per genome, when the number of DNA sample was five and sequencing depth was about 30 times (5 sample ?1 million loci ? 30 depth = 150 million). Pair-end sequencing with 100bp read length of 0.5-1 million fragments resulted in 100-200 Mb, which was estimated to represent 8-13% of the Japanese crested ibis genome, supposing that their genome size was 1.5 Gb based on several entries in the Eukaryotic genome size databases. To prepare RRL including the desired number of fragments, we digested genome DNA with HaeIII or MboI and isolated fragments in the 250-350 bp size range from agarose gel. In a preliminary experiment, the number of size selected DNA fragments by digestion with HaeIII and that by MboI were estimated to be 0.34 million and 0.44 million, respectively from the yield of isolated DNA fragments. Also, additional reason that we chose the DNA fragments in the 250-350 bp size range, was to prevent decreasing sequence data by overlapping of forward and reverse 100 bp sequence reads from a restriction fragment. The restriction fragments by digestion with HaeIII and those by MboI were combined and processed as a single RRL for sequencing. The five RRLs were independently prepared from the 5 founder genomes. Each of RRLs was distinguished by adding the sequence adaptors with different index sequence (barcode sequence). After then, the 5 RRLs were pooled and sequenced by a single sequencing lane on Hiseq2000 for 101 cycles with pair-end mode. |
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