Computational protocol: How reliably can northeast Atlantic sand lances of the genera Ammodytes and Hyperoplus be distinguished? A comparative application of morphological and molecular methods

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Protocol publication

[…] Genomic DNA was extracted at the DZMB using the Qiagen DNeasy Blood and Tissue Kit for single columns as described by . A 652-bp fragment of the mitochondrial (mt) COI gene was amplified for 38 samples using a M13 tailed primer cocktail (C_FishF1t1-C_FishR1t1) including FishF2_t1 (5’-TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC), FishR2_t1 (5’-CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA), VF2_t1 (5’-TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC) and FR1d_t1 (5’-CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA) (Ivanova et al. 2007). As a second marker, a 464 bp fragment of the nuclear (nc) Rhodpsin gene was amplified for all 70 samples using the forward primer Rh545 (5’-GCAAGCCCATCAGCAACTTCCG) and the reverse primer Rh1039r (5’-TGCTTGTTCATGCAGATGTAGA) developed by Chen et al. (2003). Each PCR reaction mixture contained 1 µl DNA template, 2.25 µl of 10X reaction buffer (including MgCl2), 0.5 µl dNTPs (2mM each), 0.5 µl of each primer (10 pmol/µl), 0.5 µl Taq polymerase (5 U/µl; Qiagen) and molecular grade water for a total volume of 25µl. Thermal cycling was performed with an initial denaturation for 2 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at the annealing temperature of 50°C, 60 s at 72°C with a final extension of 10 min at 72°C. All PCR products were checked by a 1% agarose gel. Amplicons were purified by incubating 10 µl of PCR products with 0.5 µl of Exonuclease I (20 U/µl) and 2 µl of Alkaline Phosphatase (1 U/µl) for 15 min at 37°C followed by 20 min at 75°C. Purified amplicons were sequenced by Macrogen Europe (Amsterdam, Netherlands).mitochondrial nuclear [...] Forward and reverse sequences of COI and Rhodpsin were assembled and edited using Geneious (version 7.1.9. http://www.geneious.com). Consensus sequences were submitted to GenBank (for accession numbers see Suppl. material ). Variance in sequence length, base composition, number of invariable sites and the presence of stop codons were analysed using Geneious. The nc and mt sequences were aligned independently using MUSCLE () with default settings as implemented in MEGA version 6.06 (). Primer sequences were cut from the alignment. Rhodopsin gene sequence alignment was checked by eye for species specific diagnostic characters. For COI, Kimura-2-parameter (K2P) distances were calculated in MEGA, as K2P is used as standard model for barcoding analyses and enables direct comparison with other studies. Neighbour-Joining (NJ) topology () was built in MEGA using the “pairwise deletion” option for the treatment of gaps and missing data, in order to retain all sites initially, excluding them as necessary. Node support for the NJ topology was evaluated by a non-parametric bootstrap analysis () with 10,000 replicates. In order to quantify the distinctness between species at the barcode locus, genetic distances were used to calculate the difference between the maximum intraspecific genetic distance and the minimum distance to the nearest neighbor (barcode gap). For the calculation of genetic distances at genus and family level, we used BOLDs “Distance Summary” tool by choosing K2P distance model and MUSCLE () alignment algorithm.Kimura-2-parameter Neighbour-Joining On BOLD, DNA barcodes were automatically assigned to operational taxonomic units (OTUs), generated through Refined Single Linkage (RESL) analyses (). Finally, a unique alphanumeric code is assigned to each of the OTUs, constituting the so called barcode index number (BIN). It has been shown that BINs are highly congruent with existing species assignments (). Here, the ‘BIN Discordance Report’ analysis tool was applied to analyse our dataset together with public sequences on BOLD, and to get hints on cryptic diversity (species) or to identify cases of haplotype sharing between species.operational taxonomic units Refined Single Linkage barcode index number Furthermore, BOLD’s “Diagnostic Characters” sequence analysis tool was applied to the COI dataset choosing MUSCLE () alignment algorithm. Sequences were grouped by species names in order to categorise consensus bases by their diagnostic potential. […]

Pipeline specifications

Software tools FISHR, Geneious, MUSCLE, MEGA
Applications Population genetic analysis, Nucleotide sequence alignment
Organisms Dipturus trachyderma