Miya M, Sato Y, Fukunaga T, Sado T, Poulsen JY, Sato K, Minamoto T, Yamamoto S, Yamanaka H, Araki H, Kondoh M, Iwasaki W.

MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species.

R Soc Open Sci. 2015 Jul 22;2(7):150088. doi:10.1098/rsos.150088

“Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera.”

“considering the unconventional base pairing in the T/G bond, the designed primers use G rather than A when the template is variably C or T, and T rather than C when the template is A or G;”

“2 l lots of seawater from the 10 l samples were vacuum-filtered onto 47 mm diameter glass-fibre filters [and then] stored in −20°C before eDNA extraction.” “Two litres of Milli-Q water was used as the negative control.” ”Lysis using proteinase K [at] 56°C [...] for 30 min.” “Six random hexamers (N) are used to enhance cluster separation on the flowcells during [basecall]”

“35 cycles of a 12 μl reaction volume containing 6.0 μl 2× KAPA HiFi HotStart ReadyMix (including DNA polymerase, reaction buffer, dNTPs and MgCl2 (at a final concentration of 2.5 mM)) (KAPA Biosystems, Wilmington, MA, USA), 0.7 μl of each primer (5 μM), 2.6 μl sterile distilled H2O and 2.0 μl template. [...] The thermal cycle profile after an initial 3 min denaturation at 95°C was as follows: denaturation at 98°C for 20 s; annealing at 65°C for 15 s; and extension at 72°C for 15 s with the final extension at the same temperature for 5 min.” “The first PCR product was diluted 10 times using Milli-Q water and used as a template for the second PCR.”

“The pre-processed reads from the above custom pipeline were dereplicated using a ‘derep_fulllength’ command in UCLUST”

“Optimal experimental conditions for the first PCR with these primers were achieved through trial and error, and we found that choice of a PCR kit (KAPA HiFi HotStart ReadyMix) and associated high-annealing temperatures (65–67°C) in the first PCR are the two most important factors contributing to successful amplifications showing distinct single PCR bands on the agarose gel.”

“MiFish-U/E primers also amplified eDNA from a [...] spotted dolphin”

“The occasional detection of [non-tank species] in the negative controls strongly suggests cross contamination in the laboratory, which seems unavoidable in eDNA studies using PCR amplifications.”