DOI: 10.5061/dryad.f40v5
title: Data from: A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

description: The main objective of this work was to develop and validate a robust and
 reliable ‘from benchtop-to-desktop’ metabarcoding workflow to investigate
 the diet of invertebrate-eaters. We applied our workflow to fecal DNA
 samples of an invertebrate-eating fish species. A fragment of the COI gene
 was amplified by combining two minibarcoding primer sets to maximize the
 taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq
 platform. We developed a filtering approach based on a series of
 non-arbitrary thresholds established from control samples and from
 molecular replicates in order to address the elimination of
 cross-contamination, PCR/sequencing errors and mistagging artifacts. This
 resulted in a conservative and informative metabarcoding dataset. We
 developed a taxonomic assignment procedure that combines different
 approaches and that allowed the identification of ~75% of invertebrate COI
 variants to the species level. Moreover, based on the diversity of the
 variants, we introduced a semi-quantitative statistic in our diet study,
 the Minimum Number of Individuals (MNI), which is based on the number of
 distinct variants in each sample. The metabarcoding approach described in
 this paper may guide future diet studies that aim to produce robust
 datasets associated with a fine and accurate identification of prey items.
description: COI-filtering-DB.fas.tarCustom COI database used during HTS filtering in order to keep only relevant COI sequencesTaxassign-DB.fas.tarCustom COI database used for taxonomic assignement. It contains selected COI sequences from BOLD and sequences produced in the lab (MF458551 - MF458851). Sequences are in fasta format. ID lines contain taxon names and NCBI TaxIDs. If NCBI TaxID is not available for the taxon, the TaxID of its lowest level parent is given.Perl_scripts.tarPerl script used for data filtering and taxonomic assignment.MFZR1_S4_L001_R1_001.fastq.tarFastq file with raw data. Forward sequences of the first replicate series amplified by MFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.MFZR1_S4_L001_R2_001.fastq.tarFastq file with raw data. Reverse sequences of the first replicate series amplified by MFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.MFZR2_S5_L001_R1_001.fastq.tarFastq file with raw data. Forward sequences of the second replicate series amplified by MFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.MFZR2_S5_L001_R2_001.fastq.tarFastq file with raw data. Reverse sequences of the second replicate series amplified by MFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.MFZR3_S6_L001_R1_001.fastq.tarFastq file with raw data. Forward sequences of the third replicate series amplified by MFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.MFZR3_S6_L001_R2_001.fastq.tarFastq file with raw data. Reverse sequences of the third replicate series amplified by MFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.ZFZR1_S1_L001_R1_001.fastq.tarFastq file with raw data. Forward sequences of the first replicate series amplified by ZFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.ZFZR1_S1_L001_R2_001.fastq.tarFastq file with raw data. Reverse sequences of the first replicate series amplified by ZFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.ZFZR2_S2_L001_R1_001.fastq.tarFastq file with raw data. Forward sequences of the second replicate series amplified by ZFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.ZFZR2_S2_L001_R2_001.fastq.tarFastq file with raw data. Reverse sequences of the second replicate series amplified by ZFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.ZFZR3_S3_L001_R1_001.fastq.tarFastq file with raw data. Forward sequences of the third replicate series amplified by ZFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.ZFZR3_S3_L001_R2_001.fastq.tarFastq file with raw data. Reverse sequences of the third replicate series amplified by ZFZR primer pairs. Tag combinations to identify the samples are found in the associated readme file.alignements_for_phylogeny.tarSequence alignments used for constructing phylogenetic trees of variants and their homologues.