In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets’ complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the “one‐locus‐several‐primer‐sets” (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey–predator interactions.

Villsen2023_ITV_sample_fastq_correspondence.csv

CSV file with tag combinations to identify the samples and technical replicates from each fastq file

Villsen2023_ITV _Metabarcoding_Filtered_Data.csv

CSV file reporting the metabarcoding data that past the filtering procedure. The data include ASVs and contigs, sequence data, and taxonomic assignment for Zingel asper’s feces.

Villsen2023_ITV_dietdata.csv

CSV file reporting the diet data used to estimate individual and population trophic traits of Zingel asper.
Please note that columns of Villsen2023_ITV_dietdata.csv correspond to rows in Villsen2023_ITV _Metabarcoding_Filtered_Data.csv

Villsen2023_ITV_HabitatPreydata.csv

Macroinvertebrate and habitat datasets used for statistical analysis and modelling process in Villsen2023_ITV

README_Villsen2023_ITV_HabitatPreydata.txt

List of variables in Villsen2023_ITV_HabitatPreydata.csv

 

Villsen2022_sample_fastq_correspondence.csv

CSV file with tag combinations to identify the samples and technical replicates from each fastq file

Villsen2022_Metabarcoding_Filtered_Data_040822.xlsx

CSV file reporting the metabarcoding data that past the filtering procedure. The data include ASVs and contigs, sequence data, and taxonomic assignment for Zingel asper’s feces.

Villsen2022_Diet_Data_040822.csv

CSV file reporting the diet data used to estimate individual and population trophic traits of Zingel asper.
Please note that columns of Villsen2022_Diet_Data.csv correspond to rows in Villsen2022_Metabarcoding_Filtered_Data.csv

Villsen2022_Data_For_Modelling_Analyses_040822.csv

CSV file reporting the data used for modelling analyses

Corse2019_sample_fastq_correspondence.csv

CSV file with tag combinations to identify the samples and technical replicates from each fastq files

alignments.tar

Mapping Assisted Multiple Alignment files for in silico primer testing

csv_all_runs_samples_files.csv Metadata on fastq files

Tag combinations to identify the samples and technical replicates from each fastq files

DNA1_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA1 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA1_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA1 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA1_ZFZR

Fastq file with raw data of Illumina reads Plate: DNA1 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA2_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA2 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA2_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA2 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA2_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA2 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA3_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA3 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA3_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA3 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA3_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA3 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA4_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA4 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA4_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA4 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA4_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA4 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA5_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA5 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA5_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA5 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA5_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA5 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA6_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA6 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA6_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA6 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA6_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA6 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA7_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA7 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA7_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA7 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA7_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA7 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA8_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA8 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA8_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA8 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA8_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA8 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA9_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA9 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA9_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA9 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA9_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA9 Marker: ZFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA10_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA10 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA10_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA10 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA10_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA10 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA11_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA11 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA11_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA11 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA11_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA11 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA12_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA12 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA12_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA12 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA12_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA12 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA13_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA13 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA13_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA13 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA13_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA13 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA14_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA14 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA14_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA14 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA14_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA14 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA15_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA15 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA15_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA15 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA15_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA15 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA16_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA16 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA16_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA16 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA16_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA16 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA17_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA17 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA17_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA17 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA17_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA17 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA18_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA18 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA18_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA18 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA18_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA18 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA19_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA19 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA19_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA19 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA19_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA19 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA20_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA20 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA20_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA20 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA20_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA20 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA21_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA21 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA21_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA21 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA21_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA21 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA22_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA22 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA22_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA22 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA22_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA22 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA23_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA23 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA23_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA23 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA23_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA23 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA24_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA24 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA24_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA24 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA24_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA24 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA25_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA25 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA25_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA25 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA25_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA25 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA26_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA26 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA26_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA26 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA26_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA26 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA27_LFCR.tar

Fastq file with raw data of Illumina reads Plate: DNA27 Marker: LFCR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA27_MFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA27 Marker: MFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

DNA27_ZFZR.tar

Fastq file with raw data of Illumina reads Plate: DNA27 Marker: ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'

Ext7.tar

Fastq file with raw data of Illumina reads Plate: Ext7 Marker: LFCR, MFZR, ZFZR Tag combinations to identify the samples from each fastq files are found in the 'CSV file with metadata on fastq files'