DOI: 10.5061/dryad.g14k2
title: Data from: Scrutinizing key steps for reliable metabarcoding of environmental samples

description: 1. Metabarcoding of environmental samples has many challenges and
 limitations that require carefully considered laboratory and analysis
 pipelines to ensure reliable results. We explore how decisions regarding
 study design, laboratory work and bioinformatic processing affect the
 final results, and provide guidelines for reliable study of environmental
 samples. 2. We evaluate the performance of four primer sets targeting COI
 and 16S regions characterising arthropod diversity in bat faecal samples,
 and investigate how metabarcoding results are affected by parameters
 including: i) number of PCR replicates per sample, ii) sequencing depth,
 iii) PCR replicate processing strategy (i.e. either additively, by
 combining the sequences obtained from the PCR replicates, or
 restrictively, by only retaining sequences that occur in multiple PCR
 replicates for each sample), iv) minimum copy number for sequences to be
 retained, v) chimera removal, and vi) similarity thresholds for OTU
 clustering. Lastly, we measure within- and between-taxa dissimilarities
 when using sequences from public databases to determine the most
 appropriate thresholds for OTU clustering and taxonomy assignment. 3. Our
 results show that the use of multiple primer sets reduces taxonomic biases
 and increases taxonomic coverage. Taxonomic profiles resulting from each
 primer set are principally affected by how many PCR replicates are carried
 out per sample and how sequences are filtered across them, the sequence
 copy number threshold and the OTU clustering threshold. We also report
 considerable diversity differences between PCR replicates from each
 sample. Sequencing depth increases the dissimilarity between PCR
 replicates unless the bioinformatic strategies to remove allegedly
 artefactual sequences are adjusted according to the number of analysed
 sequences. Finally, we show that the appropriate identity thresholds for
 OTU clustering and taxonomy assignment differ between target markers. 4.
 Metabarcoding of complex environmental samples ideally requires i)
 investigation of whether more than one primer sets targeting the same
 taxonomic group is needed to offset the effect of primer biases, ii) more
 than one PCR replicate per sample, iii) bioinformatic processing
 approaches of sequences that balance diversity detection with removal of
 artificial sequences, and iv) empirical selection of OTU clustering and
 taxonomy assignment thresholds tailored to each genetic marker and the
 obtained taxa.
description: Epp PCR replicate 1 -
 PE2Compressed raw sequence
 output file. Contains the following data: Epp primer set, 1st PCR
 replicate, reverse read.epp_PCR1_PE2.fastq.gzEpp PCR replicate 3 - PE2Compressed raw sequence output file. Contains the following data: Epp primer set, 3rd PCR replicate, reverse read.epp_PCR3_PE2.fastq.gzEpp PCR replicate 3 - PE1Compressed raw sequence output file. Contains the following data: Epp primer set, 3rd PCR replicate, forward read.epp_PCR3_PE1.fastq.gzEpp PCR replicate 1 - PE1Compressed raw sequence output file. Contains the following data: Epp primer set, 1st PCR replicate, forward read.epp_PCR1_PE1.fastq.gzEpp PCR replicate 2 - PE1Compressed raw sequence output file. Contains the following data: Epp primer set, 2nd PCR replicate, forward read.epp_PCR2_PE1.fastq.gzEpp PCR replicate 2 - PE2Compressed raw sequence output file. Contains the following data: Epp primer set, 2nd PCR replicate, reverse read.epp_PCR2_PE2.fastq.gzClarke PCR replicate 1 - PE2Compressed raw sequence output file. Contains the following data: Clarke primer set, 1st PCR replicate, reverse read.clarke_PCR1_PE2.fastq.gzClarke PCR replicate 1 - PE1Compressed raw sequence output file. Contains the following data: Clarke primer set, 1st PCR replicate, forward read.clarke_PCR1_PE1.fastq.gzClarke PCR replicate 2 - PE 1Compressed raw sequence output file. Contains the following data: Clarke primer set, 2nd PCR replicate, forward read.clarke_PCR2_PE1.fastq.gzClarke PCR replicate 2 - PE2Compressed raw sequence output file. Contains the following data: Clarke primer set, 2nd PCR replicate, reverse read.clarke_PCR2_PE2.fastq.gzClarke PCR replicate 3 - PE2Compressed raw sequence output file. Contains the following data: Clarke primer set, 3rd PCR replicate, reverse read.clarke_PCR3_PE2.fastq.gzClarke PCR replicate 3 - PE1Compressed raw sequence output file. Contains the following data: Clarke primer set, 3rd PCR replicate, forward read.clarke_PCR3_PE1.fastq.gzLeray PCR replicate 1 - PE1Compressed raw sequence output file. Contains the following data: Leray primer set, 1st PCR replicate, forward read.leray_PCR1_PE1.fastq.gzLeray PCR replicate 1 - PE2Compressed raw sequence output file. Contains the following data: Leray primer set, 1st PCR replicate, reverse read.leray_PCR1_PE2.fastq.gzLeray PCR replicate 2 - PE1Compressed raw sequence output file. Contains the following data: Leray primer set, 2nd PCR replicate, forward read.leray_PCR2_PE1.fastq.gzLeray PCR replicate 2 - PE2Compressed raw sequence output file. Contains the following data: Leray primer set, 2nd PCR replicate, reverse read.leray_PCR2_PE2.fastq.gzLeray PCR replicate 3 - PE1Compressed raw sequence output file. Contains the following data: Leray primer set, 3rd PCR replicate, forward read.leray_PCR3_PE1.fastq.gzLeray PCR replicate 3 - PE2Compressed raw sequence output file. Contains the following data: Leray primer set, 3rd PCR replicate, reverse read.leray_PCR3_PE2.fastq.gzZeale PCR replicate 3 - PE2Compressed raw sequence output file. Contains the following data: Zeale primer set, 3rd PCR replicate, reverse read.zeale_PCR3_PE2.fastq.gzZeale PCR replicate 3 - PE1Compressed raw sequence output file. Contains the following data: Zeale primer set, 3rd PCR replicate, forward read.zeale_PCR3_PE1.fastq.gzZeale PCR replicate 2 - PE2Compressed raw sequence output file. Contains the following data: Zeale primer set, 2nd PCR replicate, reverse read.zeale_PCR2_PE2.fastq.gzZeale PCR replicate 2 - PE1Compressed raw sequence output file. Contains the following data: Zeale primer set, 2nd PCR replicate, forward read.zeale_PCR2_PE1.fastq.gzZeale PCR replicate 1 - PE2Compressed raw sequence output file. Contains the following data: Zeale primer set, 1st PCR replicate, reverse read.zeale_PCR1_PE2.fastq.gzZeale PCR replicate 1 - PE1Compressed raw sequence output file. Contains the following data: Zeale primer set, 1st PCR replicate, forward read.zeale_PCR1_PE1.fastq.gz