Our approach relies on a single COI probe (MCSP: Metabacording by Capture with a Single Probe). We chose a COI probe (hereafter named single-probe) from a species (Danio rerio) belonging to an order (Cypriniformes) absent from our study area, the Amazon basin.
The single-probe was developed by PCR amplification of the CO subunit 1. PCR was performed with 80 ng D. rerio DNA, 0.3mM of forward and reverse [5']-Biotin-TEG primers [23 (link)] (FishF =TCA-ACC-AAC-CAC-AAA-GAC-ATT-GGC-AC , FishR = TAG-ACT-TCT-GGG-TGG-CCA-AAG-AAT-CA ), 12.5 μl HiFi PCR kit (KR0369, KAPA Biosystem), 9 μl H2O. PCR was run with the following cycling protocol: 95–3’, 98–80”, 52–40”, 72–1’- 35 cycles [24 ].The PCR product (682 bp) was purified with 1X ampure XP and then quantified.
In this study, we will compare the result from this new approach to a previous approach using four probes designed for siluriforms [21 (link)]. In the following text, we will use the term single-probe for the COI from Danio and the term siluriform probes for the four probes previously developed [21 (link)].
The single-probe was developed by PCR amplification of the CO subunit 1. PCR was performed with 80 ng D. rerio DNA, 0.3mM of forward and reverse [5']-Biotin-TEG primers [23 (link)] (FishF =
In this study, we will compare the result from this new approach to a previous approach using four probes designed for siluriforms [21 (link)]. In the following text, we will use the term single-probe for the COI from Danio and the term siluriform probes for the four probes previously developed [21 (link)].
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