DOI: 10.5061/dryad.4v9n227
title: In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

description: 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.
description: <b>MiSeq raw sequences of 12 COI & 16S minibarcodes
 from the arthropod mock community MC<sub>arthr </sub>(Run1
 part 1 to 3)</b> 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.   <b>MiSeq raw sequences
 of 12 COI & 16S minibarcodes from arthropod mock communities
 MC<sub>arthr+bat </sub>(Run1 part 2 to 3)</b>
 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 (<i>Rhinolophus ferrumequinum</i>) 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.  
 <b>MiSeq raw sequences of 12 COI & 16S minibarcodes
 from 73 negative controls (Run1 part 3 to 3)</b>
 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.   <b>MiSeq raw sequences
 of 3 COI & 16S minibarcodes from 22
 </b><b><i>Rhinolophus
 ferrumequinum</i></b><b> and <i>Myotis
 emarginatus</i> </b><b>guano samples<sub>
 </sub>(Run2 part 1 to 4)</b> 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.
   <b>MiSeq raw sequences of 3 COI
 & 16S minibarcodes from 22 </b><b><i>Rhinolophus
 ferrumequinum</i></b><b> and <i>Myotis
 emarginatus</i> </b><b>guano samples<sub>
 </sub>(Run2 part 2 to 4)</b> 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.  
 <b>MiSeq raw sequences of 3 COI minibarcodes from 22
 </b><b><i>Rhinolophus
 ferrumequinum</i></b><b> and <i>Myotis
 emarginatus</i> </b><b>guano samples<sub>
 </sub>(Run2 part 3 to 4)</b> 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.
   <b>MiSeq raw sequences of 3 COI
 minibarcodes from 22 </b><b><i>Rhinolophus
 ferrumequinum</i></b><b> and <i>Myotis
 emarginatus</i> </b><b>guano samples<sub>
 </sub>(Run2 part 4 to 4)</b> 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.  
 <b>Information concerning the samples and the positive and
 negative controls multiplexed in the MiSeq Runs 1 &
 2</b> 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.
   <b>Raw abundance tables of 12 COI
 & 16S minibarcodes from the arthropod mock communities
 MC<sub>arthr </sub>& MC<sub>arthr+bat
 </sub>(Run1)</b> 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.  
 <b>Raw abundance tables of 12 COI & 16S
 minibarcodes from 22 </b><b><i>Rhinolophus
 ferrumequinum</i></b><b> and <i>Myotis
 emarginatus</i> </b><b>guano samples<sub>
 </sub>(Run2)</b> 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.  
 <b>Reference sequences of the 33 arthropod taxa and the bat
 used in the mock communities</b> 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.  
 <b>Appendix D1. Rules used to determine the final
 identifications of OTUs in guano samples.</b>
   <b>Figure S1. Consensus sequence
 alignment of 21 arthropod orders using the PrimerMiner R
 package.</b>   <b>Figure
 D2. PCR products of the mock communities (left column) and guano samples
 (right column) on agarose gel.</b>  
 <b>Figure D3. </b><b>Picture of the DNA
 extractions on agarose gel for the 35 samples used in the two mock
 communities.</b>  
 <b>Table D1. Number of COI and 16S OTUs and sequences,
 obtained with sequences from BOLD and NCBI databases,
 respectively.</b>  
 <b>Table D2. Information about the samples, the laboratory
 controls and the technical replicates.</b>
   <b>Table D3. Mixes of dephasing
 primers used in PCR1 of the 2-step PCR including heterogeneity spacers and
 Illumina sequencing primer sequences.</b>
   <b>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).</b>
   <b>Table D5. Number of reads of the
 genuine and reference sequences of the two mock communities, for each PCR
 replicate and each primer set.</b>  
 <b>Table D6. Examples of affiliation errors for several
 primer sets in reference databases.</b>
   <b>Table D7. Mean penalty scores
 obtained for each arthropod orders (OTUs &gt; 100) with each primer
 set using the PrimerMiner program.</b>
   <b>Table D8. In silico amplification
 success obtained for each arthropod order (OTUs &gt; 100) and primer
 set using the PrimerMiner program.</b>
   <b>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 &gt; 97% and coverage &gt; 90%, ‡ identity &lt; 97%
 and/or coverage &lt;90%.</b>  
 
 <b>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. '&lt;97%' indicated a
 percentage of identity lower than 97%.</b>