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.