DOI: 10.5061/dryad.kv02g
title: Data from: Metabarcoding for the parallel identification of several hundred predators and their preys: application to bat species diet analysis

description: Assessing diet variability is of main importance to better understand the
 biology of bats and design conservation strategies. Although the advent of
 metabarcoding has facilitated such analyses, this approach does not come
 without challenges. Biases may occur throughout the whole experiment, from
 fieldwork to biostatistics, resulting in the detection of false negatives,
 false positives or low taxonomic resolution. We detail a rigorous
 metabarcoding approach based on a short COI minibarcode and two-step PCR
 protocol enabling the ‘all at once’ taxonomic identification of bats and
 their arthropod preys for several hundreds of samples. Our study includes
 faecal pellets collected in France from 357 bats representing 16 species,
 as well as insect mock communities that mimic bat meals of known
 composition, negative and positive controls. All samples were analysed
 using three replicates. We compare the efficiency of DNA extraction
 methods and we evaluate the effectiveness of our protocol using
 identification success, taxonomic resolution, sensitivity, and
 amplification biases. Our parallel identification strategy of predators
 and preys reduces the risk of mis-assigning preys to wrong predators and
 decreases the number of molecular steps. Controls and replicates enable to
 filter the data and limit the risk of false positives, hence guaranteeing
 high confidence results for both prey occurrence and bat species
 identification. We validate 551 COI variants from arthropod including 18
 orders, 117 family, 282 genus and 290 species. Our method therefore
 provides a rapid, resolutive and cost-effective screening tool for
 addressing evolutionary ecological issues or developing
 ‘chirosurveillance’ and conservation strategies.
description: MiSeq raw sequences of
 the COI minibarcode from the faecal pellets of 357 bats from Western
 France (part 1 to 2)This ZIP file contains
 the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2)
 produced for each faecal pellet in triplicate using the MiSeq platform.
 The 1162 multiplexed PCR products were indexed using both forward and
 reverse indices. The list of the 357 multiplexed samples and the 24
 positive and 58 negative controls are provided in the following XLSX file
 titled: Information concerning the samples multiplexed in the MiSeq
 Run.MiSeq_Reads_COI_Bat_
 faecal_samples_part1.zipMiSeq raw sequences of
 the COI minibarcode from the faecal pellets of 357 bats from Western
 France (part 2 to 2)This ZIP file contains
 the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2)
 produced for each faecal pellet in triplicate using the MiSeq platform.
 The 1162 multiplexed PCR products were indexed using both forward and
 reverse indices. The list of the 357 multiplexed samples and the 24
 positive and 58 negative controls are provided in the following XLSX file
 titled: Information concerning the samples multiplexed in the MiSeq
 Run.MiSeq_Reads_COI_Bat_
 faecal_samples_part2.zipMiSeq raw sequences of
 the COI minibarcode from 24 positive controls and 58 negative
 controlsThis ZIP file contains
 the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2)
 produced for each faecal pellet in triplicate using the MiSeq platform.
 The 1162 multiplexed PCR products were indexed using both forward and
 reverse indices. The list of the 357 multiplexed samples and the 24
 positive and 58 negative controls are provided in the following XLSX file
 titled: Information concerning the samples multiplexed in the MiSeq
 Run.MiSeq_Reads_COI_Controls.zipMiSeq raw sequences of the COI minibarcode from arthropod mock communities MC1 & MC2This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced using the MiSeq platform for each individual PCR and the pools of the arthropod mock communities in triplicate. The 1162 multiplexed PCR products were indexed using both forward and reverse indices. The list of the 357 multiplexed samples and the 24 positive and 58 negative controls are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Run.MiSeq_Reads_COI_Mock_communities.zipRaw output files generated by the mothur programThis ZIP file contains three output files (.table, .fasta and .log) generated by the mothur program for the whole COI minibarcode dataset. The .log file contains all the mothur command lines used for the processing of the raw sequences.mothur_raw_output_files.zipInformation concerning the samples and positive and negative controls multiplexed in the MiSeq runThis XLSX file contains the sample IDs, the sample types, the PCR IDs, the PCR replicate numbers, the localities, the bat species and the fastq file names for each of the 1162 PCR products multiplexed in the Illumina MiSeq run.Information_concerning_the samples.xlsxRaw abundance table before filteringThis XLSX file contains the number of reads for each distinct variant of the MiSeq run and for each PCR product before the data filtering process.Table_of_abundance_before_filtering.xlsxAbundance table after filteringThis XLSX file contains the number of reads for each distinct variant of the MiSeq runs and each sample after the data filtering. The results for the three PCR replicates of a same sample were summed. The sheet "Bat faecal samples" contains the results for the faecal pellets from bats, and the sheet "Mock communities" contains the results for the arthropod mock communities.Table_of_abundance_after_filtering.xlsxAlignment of 133bp minibarcode COI reference sequences from Barcode of Life Database and corresponding to 33 bat species found in FranceThis FASTA file contains 444 reference sequences available in BOLD for the 33 bat species found in France. We removed sequences showing less than 132bp for the COI minibarcode used in our study and/or showing one or more ambiguous base (N). Note that the sequences GBMA9839-15, ABBWP228-07, NOMAM103-16, NOMAM142-16 and SKBPA621-11 might not be reliable: they could have been assigned to the wrong species or may be of low quality (see results in the phylogenetic tree and Table S4, Supplemental Information).Alignement_COI_133bp_minibarcode_444_BOLD_reference_sequences.fasta.txtAlignment of COI reference sequences available for 33 bat species found in France and recorded in Barcode of Life Database for the 29bp position of the reverse primerThis FASTA file contains 693 reference sequences from BOLD corresponding to the 33 bat species found in France. We removed sequences showing less than 29bp for the position of the reverse primer used in our study and/or showing one or more ambiguous base (N). See also Table S4 for a summary.Alignement_COI_29bp_revers primer_693_BOLD_reference_sequences.fastaAlignment of the COI haplotypes obtained by Sanger and MiSeq sequencing for the 19 insect species included in the mock communitiesThis FASTA file contains 19 and 20 COI sequences generated by Sanger and Illumina Miseq sequencing respectively. The Sanger sequences correspond to a fragment of 634 to 658bp of the standard COI barcode following the Barcode of Life Consortium. The MiSeq sequences correspond to the 133bp COI minibarcode used in this study. For this latter, only the most abundant variant for each of the 19 samples is provided, except for Forficula lesnei specimen for which an unexpected parasitoid (Triarthria setipennis) was detected and is also provided.Alignment_COI_Sanger_MiSeq_Mock_Communities.fasta