Computational protocol: Habitat suitability and movement corridors of grey wolf (Canis lupus) in Northern Pakistan

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Protocol publication

[…] Deoxyribonucleic acid (DNA) was extracted from 15 mg of feces using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) recovered in a total volume of 200 μL. Blank extractions were systematically performed to monitor possible contaminations. Species identification was performed through next generation sequencings (NGS) by amplifying DNA extract using primer pair 12SV5F (5’-TAGAACAGGCTCCTCTAG-3’) and 12SV5R (5’- TTAGATACCCCACTATGC-3’) [] targeting about 100-bp of the V5 loop of the mitochondrial 12S gene []. The sequencing was carried out on the Illumina Genome Analyzer IIx (Illumina Inc.), using the Paired-End Cluster Generation Kit V4 and the Sequencing Kit V4 (Illumina Inc.), following the manufacturer’s instructions. The sequence reads were analyzed using OBI Tools ( Taxon assignation was achieved using the ecoTag program [] in comparison with a reference database for vertebrates. This reference database was built by extracting the relevant part of the mitochondrial 12S gene from the European Molecular Biology Laboratory’s (EMBL) nucleotide library using the ecoPCR program []. Genetic results revealed identification of 80 samples belonging to wolves. […]

Pipeline specifications

Software tools OBITools, ecoPCR
Application qPCR