DOI: 10.5285/3affed0d-fe6f-4916-89e3-e672639191e5
title: Mammal occurrence identified from metabarcoding of two leech species from Sabah, Malaysia

description: This dataset contains the results from a metabarcoding study of terrestrial leech blood meals to detect differences in the diets of two leech species, Haemadipsa picta and Haemadipsa sumatrana. Mammal taxa were identified using metabarcoding of 16s rRNA and comparisons of operational taxonomic units (OTUs) to a curated reference database from NCBI (National Center for Biotechnology Information) GenBank. All leeches were collected from the Stability of Altered Forest Ecosystems project (SAFE; www.SAFEproject.net) as part of the NERC Human Modified tropical Forest Programme and the LOMBOK consortia (Land-use Options for Maintaining BiOdiversity & eKosystem functions). Leech samples were collected at different sites across a habitat gradient, to assess these invertebrates as molecular sampling tools for mammals. Individuals were pooled before amplicon sequencing with Illumina MiSeq 150-200bp x2. The resultant raw sequences were filtered and clustered at 97%, curated and then assigned to the reference database using BLAST and MEGAN programmes. 
description: Individuals of both species were collected at 25m2 vegetation plots in the forest blocks (A-F, LFE, LF1-3 & VJR) at the SAFE project Sabah (corresponding to second order points). Plots are approximately 173m apart and all plots were sampled 4 times between February 2015 and June 2015. Sampling consists of searching the leaf litter and undergrowth for leeches for a fixed time of 20 minutes. Individuals are kept in RNALater to stabilise DNA until extraction. DNA extraction of pools of ten individual leeches was undertaken using a digestion buffer with modified QiaQuick (Qiagen) protocol and dual tagged PCR in triplicate reactions (details in Drinkwater et al. in review).  After screening samples for successful PCR amplification, a total of 41 samples of pooled Haemadipsa sumatrana and 49 pooled Haemadipsa picta from six sites remained for sequencing (B, F, D, LF, LFE, VJR). Amplicons were pooled for library build following Carøe et al, 2017) and sequencing with Illumina MiSeq 150-200bp x2. Sumaclust was used for OTU clustering (Mercier et al., 2013) and LULU was used for post-clustering filtering (Frøslev et al., 2017). This resulted in the final contingency table comparing mammal detections with leech pool. 

Carøe, C., Gopalakrishnan, S., Vinner, L., Mak, S. S. T., Sinding, M. H. S., Samaniego, J. A., Wales, N., Sicheritz-Ponten, M., Gilbert, M. T. P. (2017). Single-tube library preparation for degraded DNA. Methods in Ecology and Evolution, 9(2), 410-419. https://doi.org/10.1111/2041-210X.12871

Frøslev, T. G., Kjøller, R., Bruun, H. H., Ejrnæs, R., Brunbjerg, A. K., Pietroni, C., & Hansen, A. J. (2017). Algorithm for post-clustering curation of DNA amplicon data yields reliable biodiversity estimates. Nature Communications, 8(1), 1188. https://doi.org/10.1038/s41467-017-01312-x

Mercier, C., Boyer, F., Bonin, A., & Coissac, E. (2013). SUMATRA and SUMACLUST: fast and exact comparison and clustering of sequences. In Programs and Abstracts of the SeqBio workshop (pp. 27-29).