DOI: 10.5061/dryad.44j0zpcfp
title: Sequencing data and taxonomic assignments from: Biodiversity and vector-borne diseases: host dilution and vector amplification occur simultaneously for Amazonian leishmaniases

description: This is the sequencing data used in the paper: "Biodiversity and
 vector-borne diseases: host dilution and vector amplification occur
 simultaneously for Amazonian leishmaniases" by Kocher et al. The
 study aims at assessing the effects of biodiversity changes on Leishmania
 transmission using molecular analyses of sand fly pools and blood-fed
 dipterans. The data is split in three files corresponding to the PCR
 amplicons used in the study (Ins16S for insect identifications, 12SV5 for
 vertebrate identifications and leishmini for Leishmania identifications).
 Each file combines output from different Illumina Miseq and Hiseq runs,
 after read demultiplexing and adapter trimming, dereplication and removal
 of reads present in less than 10 copies (but before further read
 filtering). The data is presented in tabular format (similar to the output
 of the obitab command from the obitools package), together with
 information on the corresponging sample, sequencing run, and taxonomic
 assignments (performed with ecotag from the obitools).
description: (See the paper for more details) Insects were
 sampled between 2015 and 2017 in French Guiana using Centre for Disease
 Control light traps. Each morning, the content of each trap was collected.
 Sand fly females were sorted using a stereo microscope and kept in pools
 (corresponding to each trap-night) in microcentrifuge tubes with 95%
 ethanol for later molecular analyses. A maximum of 50 individuals was
 included in a pool, and several pools were made when more than 50
 specimens were caught in a given trap (with a maximum of four pools per
 trap, i.e. 200 individuals). Visibly blood-fed dipterans, including sand
 flies, mosquitoes (Culicidae), and biting midges (Ceratopogonidae), were
 kept individually in microcentrifuge tubes with 95% ethanol for molecular
 analyses. Additional blood-fed dipterans resting during the day along tree
 trunks were collected using a Prokopack aspirator (John W. Hock co.,
 Gainesville, FL, USA) and conserved in the same way. We
 analyzed sand fly pools to identify their species composition using DNA
 metabarcoding. Leishmania DNA detection and identification was performed
 on the same pools using high-throughput sequencing of kDNA minicircle
 amplicons. For each blood-fed specimen, the dipteran species and blood
 meal source were also identified molecularly. Sand fly pools were
 homogenized using a Qiagen TissueLyser 2 (Qiagen, Valencia, CA, USA), and
 DNA was extracted with the Qiagen DNeasy Blood and Tissue kit. For
 individual blood-fed specimens, a modified Chelex (Bio-Rad, Hercules, CA,
 USA) protocol was used for DNA extraction. The Ins16S_1 [F:
 TRRGACGAGAAGACCCTATA; R: TCTTAATCCAACATCGAGGTC; (Clarke, Soubrier,
 Weyrich, & Cooper, 2014)], 12S-V5 [F: TAGAACAGGCTCCTCTAG; R:
 TTAGATACCCCACTATGC; (Riaz et al., 2011)] and leishmini [F:
 5′-GGKAGGGGCGTTCTGC-3′; R: 5′-STATWTTACACCAACCCC-3′; Kocher, Valière,
 Bañuls, & Murienne, 2018] PCR primers were used to amplify short
 fragments of dipteran, vertebrate and Leishmania DNA respectively. Tags of
 eight base pairs with at least five differences between them were added at
 the 5’ end of each primer to enable multiplexing of PCR products for
 subsequent sequencing. A Latin square design was used for PCR multiplexing
 to allow for the detection and filtering of mistagged sequencing read. For
 sand fly metabarcoding, two PCR replicates were performed. PCR products
 were pooled according to the multiplexing design and used for sequencing
 library preparation and high-throughput sequencing on Illumina Hiseq or
 Miseq platforms at the GeT-PlaGe core facilities of Genotoul (Toulouse,
 France). Bioinformatic analyses were performed using
 the OBITools 1.2.9 package and R 4.0.3. Paired-end reads were merged with
 illuminapairedend and demultiplexed based on PCR primer tags using
 ngsfilter. Reads were dereplicated using obiuniq and sequences supported
 by less than ten reads in a given sample were discarded using obigrep.
 Taxonomic assignments were performed using ecotag, with reference DNA
 sequence datasets constituted for each studied taxonomic group. For
 dipteran identifications, we used previously published reference datasets
 for neotropical sand flies and mosquitoes, to which we added mosquito
 reference sequences corresponding to the targeted 16S region, which we
 extracted from NCBI GenBank using ecoPCR. For vertebrate identifications,
 we used a previously published reference dataset for Amazonian mammals,
 completed with vertebrate reference sequences corresponding to the
 targeted 12S region extracted from GenBank. For Leishmania
 identifications, we use a previously published dataset of kDNA minicircle
 reference sequences. We considered taxonomic assignments at the genus
 level at best if the percentage of identity with the closest match was
 lower than 97%, in order to avoid biases due to reference dataset
 incompleteness (i.e. artifactual species-level identifications in cases
 where only one species was represented in the dataset for a given genus).
 We then performed de novo sequence clustering using sumaclust 1.0.31, with
 a 97% threshold. We defined molecular taxonomic units (MOTU) based on
 ecotag results in case of species-level identifications, and based on de
 novo clustering otherwise, in order to identify putative species within
 upper-level taxa. Vertebrate identifications were adjusted when only a
 subset of the matched species was known to be present in French Guiana.
 Because no reference sequence was available for local biting midge
 species, we defined MOTUs within the Ceratopogonidae family based on de
 novo clustering only.