Computational protocol: Potential DNA barcodes for Melilotus species based on five single loci and their combinations

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Protocol publication

[…] Contigs were assembled and edited prior to alignment. The Contig Express module of Vector NTI Suite 6.0 software was used to remove both ends of the sequences and to keep the head and tail of the same gene at homologous sites. Sequences were aligned using DNAMAN6.0. [...] Sequence alignment for the five DNA regions was performed using ClustalW of MEGA 6.0 software []. The Neighbour-Joining (NJ) method was used to generate a phylogenetic tree to obtain a pre-estimate of the discrimination ability of the five barcodes. The number of differences was used, and bootstrap values were calculated for 1000 replicates during construction of the NJ tree. Inter-specific genetic pairwise distances were calculated by Computing Pairwise Distance using MEGA 6.0 software. The candidate barcodes were classified on the basis of their identification ability. The sequences for each potential barcode were aligned among pairs, and the Emboss Needle algorithm ( was used to calculate the barcode gap value, score and similarity of each sequence. For comparison of gaps between accessions, 36 datasets were divided into two subsets according to species affinity, resulting in 18 sequences representing 18 species in each group. […]

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