DNA quality and quantity
Long fragments of DNA were extracted suggesting very little fragmentation of the DNA with the phenol-chloroform and phase lock method for the fresh O. tshawytscha specimen (DIN>9 for gDNA), apart from the fragmentation caused by the transposase activity of the rapid library protocol and the targeted scission of the Cas9 nuclease. Among all the sequences obtained, a great number of complete mitogenome sequences were observed that were simply linearized by the Cas9 or the transposase enzymes. Slightly lower DIN (8.9) was obtained for T. pacificus that were kept at -80°C for approximately a month and transported in dry ice for 6 h. Even lower values (<6.5) were obtained for M. pacificus, T. crenularis, D. theta and S. leucopsarus that were kept at -80°C for a month but transported with ice blocks for 18h. Almost complete degradation of the DNA (DIN<2) was observed on a T. pacificus samples that had been always kept at -80°C for more than a year (Table 2, S2).
The mitoenrichment method augmented the ratio of mitochondrial DNA from 2-fold in the case of heart to 26-fold for skeletal muscle (Table 2) and preserved the DNA intact with DIN~7. Heart and liver produced more mtDNA than muscle, respectively, but also more nDNA and hence the increment of the proportion of target to total DNA is not as pronounced as muscle mitochondrial isolations (Table 2). Liver was not useful in combination with Longmire buffer for the extraction of gDNA because the sample coagulated and made the subsequent extraction steps difficult and an unattainable eluate to work with because the DNA extract was not clean and difficult to pipette. Moreover, the subsequent sequencing run produced very few sequences compared to other runs with no clear size pattern despite having great amounts of DNA and thus liver as a tissue for genomic DNA extraction with Longmire buffer was discarded. However, useful mtDNA was isolated from liver for chinook.
Table 2 DNA extraction comparison by species, tissue type, DNA extraction and integrity (DIN), total DNA quantity, target DNA copy number, target DNA concentration and ratio of target-to-total DNA.gDNA: genomic DNA extraction, mtDNA: mitochondria selection and DNA extraction. The target DNA concentration (only explored for O. tshawytscha ) is in ng ul-1 and target ratio are calculated assuming intact mtDNA of 16,633 bp.