MiSeq raw sequences of 12 COI & 16S minibarcodes from the arthropod mock community MCarthr (Run1 part 1 to 3) This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for a pool of 33 arthropod taxa amplified using 12 COI & 16S primer sets and sequenced in triplicate using the MiSeq platform. These 36 multiplexed PCR products were indexed using both forward and reverse indexes. The list of these 36 multiplexed samples and the other samples and negative controls of this sequencing run are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Runs. MiSeq raw sequences of 12 COI & 16S minibarcodes from arthropod mock communities MCarthr+bat (Run1 part 2 to 3) This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for a pool of 33 arthropod taxa + 1 chiroptera taxon (Rhinolophus ferrumequinum) amplified using 12 COI & 16S primer sets and sequenced in triplicate using the MiSeq platform. These 36 multiplexed PCR products were indexed using both forward and reverse indexes. The list of these 36 multiplexed samples and the other samples and negative controls of this sequencing run are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Runs. MiSeq raw sequences of 12 COI & 16S minibarcodes from 73 negative controls (Run1 part 3 to 3) This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for 73 negative controls (extraction, PCR & indexing) amplified using 12 COI & 16S primer sets and sequenced using the MiSeq platform. The negative controls for extractions and PCRs were indexed using both forward and reverse indexes. The list of these 73 negative controls negative controls and the other samples of this sequencing run are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Runs. MiSeq raw sequences of 3 COI & 16S minibarcodes from 22 Rhinolophus ferrumequinum and Myotis emarginatus guano samples (Run2 part 1 to 4) This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for 22 guano samples amplified using two COI (MG2 & fwh2) and one 16S (Epp-degen) primer sets and sequenced in triplicate using the MiSeq platform. These 87 multiplexed PCR products, including positive and negative controls, were indexed using both forward and reverse indexes. The same index pair was used for the three different minibarcodes from a same sample and technical replicate. The list of these 87 multiplexed samples and the other samples and negative controls of this sequencing run are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Runs. MiSeq raw sequences of 3 COI & 16S minibarcodes from 22 Rhinolophus ferrumequinum and Myotis emarginatus guano samples (Run2 part 2 to 4) This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for 22 guano samples amplified using two COI (MG2fwh & mlHCO) and one 16S (Epp) primer sets and sequenced in triplicate using the MiSeq platform. These 81 multiplexed PCR products, including positive and negative controls, were indexed using both forward and reverse indexes. The same index pair was used for the three different minibarcodes from a same sample and technical replicate. The list of these 81 multiplexed samples and the other samples and negative controls of this sequencing run are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Runs. MiSeq raw sequences of 3 COI minibarcodes from 22 Rhinolophus ferrumequinum and Myotis emarginatus guano samples (Run2 part 3 to 4) This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for 22 guano samples amplified using three COI primer sets (MG2-ANMLdegen, Lep1 & Zeale) and sequenced in triplicate using the MiSeq platform. These 81 multiplexed PCR products, including positive and negative controls, were indexed using both forward and reverse indexes. The same index pair was used for the three different minibarcodes from a same sample and technical replicate. The list of these 81 multiplexed samples and the other samples and negative controls of this sequencing run are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Runs. MiSeq raw sequences of 3 COI minibarcodes from 22 Rhinolophus ferrumequinum and Myotis emarginatus guano samples (Run2 part 4 to 4) This ZIP file contains the FASTQ files of the paired-end reads (R1: reads 1; R2: reads 2) produced for 22 guano samples amplified using three COI primer sets (fwh1, fwhFol & HEX) and sequenced in triplicate using the MiSeq platform. These 81 multiplexed PCR products, including positive and negative controls, were indexed using both forward and reverse indexes. The same index pair was used for the three different minibarcodes from a same sample and technical replicate. The list of these 81 multiplexed samples and the other samples and negative controls of this sequencing run are provided in the following XLSX file titled: Information concerning the samples multiplexed in the MiSeq Runs. Information concerning the samples and the positive and negative controls multiplexed in the MiSeq Runs 1 & 2 This XLSX file contains the run names, the sample names, the dual-index pairs, the sample types, the PCR IDs, the PCR replicate numbers and the fastq file names for each of the 1045 PCR products multiplexed in two different Illumina MiSeq runs. Raw abundance tables of 12 COI & 16S minibarcodes from the arthropod mock communities MCarthr & MCarthr+bat (Run1) This ZIP file contains 12 TSV files (one for each minibarcode) with the number of reads for each distinct variant and each PCR product of the mock communities and controls sequenced in the MiSeq Run 1. Raw abundance tables of 12 COI & 16S minibarcodes from 22 Rhinolophus ferrumequinum and Myotis emarginatus guano samples (Run2) This ZIP file contains 12 TSV files (one for each minibarcode) with the number of reads for each distinct variant and each PCR product of the 22 guano samples and controls sequenced in the MiSeq Run 2. For the COI data, the 12 multi-hits files containing the list of the multi-affiliations for the concerning variants are also provided. For the 16S data, the 2 taxonomic files generated by the BLASTn program are also provided. Reference sequences of the 33 arthropod taxa and the bat used in the mock communities This FASTA file contains 417 sequences including (1) 35 Sanger sequences of the 33 arthropod taxa and the bat used to build the mock communities and (2) 382 sequences of the genuine variants obtained by MiSeq sequencing of the mock communities for the 12 minibarcodes. Appendix D1. Rules used to determine the final identifications of OTUs in guano samples. Figure S1. Consensus sequence alignment of 21 arthropod orders using the PrimerMiner R package. Figure D2. PCR products of the mock communities (left column) and guano samples (right column) on agarose gel. Figure D3. Picture of the DNA extractions on agarose gel for the 35 samples used in the two mock communities. Table D1. Number of COI and 16S OTUs and sequences, obtained with sequences from BOLD and NCBI databases, respectively. Table D2. Information about the samples, the laboratory controls and the technical replicates. Table D3. Mixes of dephasing primers used in PCR1 of the 2-step PCR including heterogeneity spacers and Illumina sequencing primer sequences. Table D4. Information about the sequencing libraries derived from the samples, the laboratory controls and the technical replicates. Error-proof indexes for high throughput sequencing were created by Martin (2019). Table D5. Number of reads of the genuine and reference sequences of the two mock communities, for each PCR replicate and each primer set. Table D6. Examples of affiliation errors for several primer sets in reference databases. Table D7. Mean penalty scores obtained for each arthropod orders (OTUs > 100) with each primer set using the PrimerMiner program. Table D8. In silico amplification success obtained for each arthropod order (OTUs > 100) and primer set using the PrimerMiner program. Table D9. Objectives and impact of the pre-process and FROGS pipelines on the number of reads. ¶ 'Complete run ' indicates that the run included samplesother than those of the two mock communities. ¥ = genuine sequences, † = identity > 97% and coverage > 90%, ‡ identity < 97% and/or coverage <90%. Table D10. Molecular identification of the taxa in the mock communities obtained using the minibarcode sequences of each specimen, respectively for each primer set and the Folmer region. '<97%' indicated a percentage of identity lower than 97%.

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo-mock community (33 arthropod taxa from 16 orders), and guano-based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species which live together in mixed-colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy’s bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano-based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers’ capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.