Multiplexed sequencing files and bioinformatic pipeline for "Filtration of airborne eDNA outperforms leaf swabs for terrestrial vertebrate detection in a temperate mixed forest with longer filtration times performing the best"

These files include the Multiplexed sequencing files and bioinformatic pipeline for the manuscript entitled “Filtration of airborne eDNA outperforms leaf swabs for terrestrial vertebrate detection in a temperate mixed forest with longer filtration times performing the best”. For each sample, including blanks and positive controls, six independent PCR replicates were performed. Sample-specific barcoding was introduced during the first PCR using 7 bp tags attached to the 5′ ends of both forward and reverse primers (Bohmann et al. 2022). Unique combinations of forward and reverse tags were used for each PCR replicate and were not reused within a replicate set. This design allowed PCR products to be pooled immediately after the first PCR step. Illumina libraries were subsequently constructed from the purified amplicon pools using the PCR-free TagSteady dual-indexing protocol (Carøe and Bohmann 2020). Libraries were sequenced on ca. 27 % of an Illumina NovaSeq 6000 SP lane (XP, v1.5, 250 bp paired-end) at the GeoGenetics Sequencing Core, University of Copenhagen, Denmark. The sequence data are provided per amplicon pool, and sample demultiplexing can be performed using the accompanying “XX_Tags.txt” file. Files and descriptions - Each folder contains sequences and bioinformatic pipeline for the two primer sets (16Smam - a primer pair targeting mitochondrial 16S region of mammals with an appx. 90 bp amplicon size and BirT - a primer pair targeting the mitochondrial 12S region of birds with an appx. 260 bp amplicon length). - In each folder there are 6 types of files. 1. Bioinformatic script: .sh files contain the script that was used to analyse sequences (Begum metabarcoding pipeline - Yang et al. 2021). 2. Primers.txt: forward and reverse primer sequences. 3. PoolInfo.txt: The directory of the merged pools. NOTE: The directory will depend on the users’ working directory. 4. Fastq.gz files: Paird-end Multiplexed sequences of each pool (R1 and R2). 5. Tags.txt: 7 bp barcodes used for tagging samples. 6. PSinfo.txt: The specific tag (barcode) combinations used for labelling each sample and their corresponding sequencing pools to use during the demultiplexing. In addition to samples there are POS (positives), Filt_bl (filter blanks), EC (extraction blanks), and RC (room controls). References Bohmann, K. et al. Strategies for sample labelling and library preparation in DNA metabarcoding studies. Mol. Ecol. Resour. 22, 1231-1246 (2022). Carøe, C. & Bohmann, K. Tagsteady: A metabarcoding library preparation protocol to avoid false assignment of sequences to samples. Mol. Ecol. Resour. 20, 1620–1631 (2020). Yang, C. et al. Biodiversity Soup II: A bulk‐sample metabarcoding pipeline emphasizing error reduction. Methods Ecol. Evol. 12, 1252–1264 (2021).