Computational protocol: Population genetic structure of the land snail Camaena cicatricosa (Stylommatophora, Camaenidae) in China inferred from mitochondrial genes and ITS2 sequences

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Protocol publication

[…] Approximately 0.02–0.04 g of foot muscle tissue was used for DNA extraction. The muscle tissue was bathed in sterile water for 3–6 hours to remove residual alcohol. Total genomic DNA was isolated using the Qiagen DNeasy Blood and Tissue kit (QIAGEN), and stored at −20 °C for further use. Partial fragments of the mitochondrial COI and 16srRNA genes, and the total sequence of ITS2 were amplified via PCR using the primer pairs (Table ), reaction systems and amplification conditions listed in Table . The PCR products were analyzed through 1.2% agarose gel electrophoresis.After sequencing, the raw sequences were proof-read in chromatograms and aligned into contigs using BioEdit 7.2. ITS2 sequences were annotated using HMMer and the ITS2 Database. The alignment of mitochondrial protein-coding genes was inferred from the amino acid alignment and examined for the presence of stop codons and other indicators. Sequence alignments were generated using ClustalW implemented in MEGA 6. Sixty-seven haplotype sequences were generated and deposited in GenBank under accession numbers KU927017–KU927046 for COI, KX365248–KX365255 for 16srRNA and KU958515–KU958543 for ITS2.The nucleotide composition, mutation sites and base substitution were analyzed in MEGA 6. The number of polymorphic sites (S), haplotype diversity (Hd), nucleotide diversity (Pi), average number of nucleotide differences (K) and number of haplotypes (Hap) were calculated using DnaSP 5.0 or Arlequin 3.5. The gene flow was estimated using DnaSP 5.0 too.The genetic differentiation among populations was further estimated based on pairwise FST values (the values of fixation indices of genetic differentiation between populations). Arlequin 3.5 was employed to calculate the genetic distance matrix to test for the presence of IBD in the dataset. Google Earth ( was used to estimate the linear geographic distance (km) between the sampling locations. Significance was tested with the Mantel test employing 1000 randomizations in IBDWS 3.23. […]

Pipeline specifications

Software tools BioEdit, HMMER, Clustal W, MEGA, DnaSP, Arlequin, IBDWS
Databases ITS2 Database
Applications Phylogenetics, Population genetic analysis
Organisms Camaena cicatricosa, Angiostrongylus cantonensis, Homo sapiens
Chemicals Nucleotides