DOI: 10.17044/scilifelab.27202368.v1
title: Processed ASV data from the Insect Biome Atlas Project

description: The Insect Biome Atlas project was supported by the Knut and Alice Wallenberg Foundation (dnr 2017.0088). The project analyzed the insect faunas of Sweden and Madagascar, and their associated microbiomes, mainly using DNA metabarcoding of Malaise trap samples collected in 2019 (Sweden) or 2019–2020 (Madagascar).
Please cite this version of the dataset as:Miraldo A, Iwaszkiewicz-Eggebrecht E, Sundh J, Lokeshwaran M, Granqvist E, Goodsell R, Andersson AF, Lukasik P, Roslin T, Tack A, Ronquist F. 2024. Processed ASV data from the Insect Biome Atlas Project, version 1. doi:10.17044/scilifelab.27202368.v1 or https://doi.org/10.17044/scilifelab.27202368.v1
This dataset contains the results from bioinformatic processing of version 1 of the amplicon sequence variant (ASV) data from the Insect Biome Atlas project, that is, the CO1 metabarcoding data from Malaise trap samples processed using the FAVIS mild lysis protocol (Iwaszkiewicz et al. 2023). The bioinformatic processing involved: (1) taxonomic assignment of ASVs, (2) chimera removal; (3) clustering into OTUs; and (4) noise filtering. The clustering step involved resolution of the taxonomic annotation of the cluster and identification of a representative ASV. The noise filtering step involved removal of ASV clusters identified as potentially originating from nuclear mitochondrial DNA (NUMTs) or representing other types of error or noise. ASV taxonomic assignments, ASV cluster designations, consensus taxonomies, sequences of cluster representatives and summed counts of clusters in the sequenced samples are provided in compressed tab-separated files.The bioinformatic processing pipeline is further described in Sundh et al. (2024). <strong>NB! All result files include ASVs and clusters that represent biological spike-ins.</strong>
Methods
Taxonomic assignment
ASVs were taxonomically assigned using kmer-based methods implemented in a Snakemake workflow available here. Specifically ASVs were assigned a taxonomy using the SINTAX algorithm in vsearch (v2.21.2) using a CO1 database constructed from the Barcode Of Life Data System (Sundh 2022).
Chimera removal
The workflow first identifies chimeric ASVs in the input data using the ‘uchime_denovo’ method implemented in vsearch. This was done with a so-called ‘strict samplewise’ strategy where each sample was analysed separately (hence the ‘samplewise’ notation), only comparing ASVs present in the same sample. Further, ASVs had to be identified as chimeric in all samples where they were present (corresponding to the ‘strict’ notation) in order to be removed as chimeric.
ASV clustering
Non-chimeric sequences were then split by family-level taxonomic assignments and ASVs within each family were clustered in parallel using swarm (v3.1.0) with differences=13. Representative ASVs were selected for each generated cluster by taking the ASV with the highest relative abundance across all samples in a cluster. Counts were generated at the cluster level by summing over all ASVs in each cluster.
Consensus taxonomy
A consensus taxonomy was created for each cluster by taking into account the taxonomic assignments of all ASVs in a cluster as well as the total abundance of ASVs. For each cluster, starting at the most resolved taxonomic level, each unique taxonomic assignment was weighted by the sum of read counts of ASVs with that assignment. If a single weighted assignment made up 80% or more of all weighted assignments at that rank, that taxonomy was propagated to the ASV cluster, including parent rank assignments. If no taxonomic assignment was above the 80% threshold, the algorithm continued to the parent rank in the taxonomy. Taxonomic assignments at any available child ranks were set to the consensus assignment prefixed with ‘unresolved’.
Noise filtering and cleaning
ASV clusters were further analyzed to remove clusters likely to originate from nuclear mitochondrial DNA (NUMTs) or to represent other types of errors or noise, using an algorithm based on abundance, negative controls and taxonomic annotation uncertainty. Specifically, we removed clusters for which we only had one or two reads. For Swedish data, we also removed clusters that were not successfully assigned to at least family level by SINTAX; according to several tests, this procedure removed potential NUMTs with good accuracy (Sundh et al. 2024). For the Madagascar data, the SINTAX annotation often failed at the family level, indicating that the taxonomy annotation filtering flagged too many clusters as potential NUMTs. We therefore filtered the Madagascar data only based on the number of reads.<br>
As a last clean-up step in the noise filtering, clusters containing at least one ASV present in more than 5% of blanks were removed.
<br>
The chimera filtering and ASV clustering methods have been implemented in a Snakemake workflow available at https://github.com/insect-biome-atlas/happ. This workflow takes as input:

 The ASV sequences in FASTA format
 A tab-delimited file of counts of ASVs (rows) in samples (columns)
 Taxonomic assignments of ASVs

Data for 1) and 2) are available at https://doi.org/10.17044/scilifelab.25480681.v1, and 3) is available in this upload.
Noise filtering and cleaning was done with custom python scripts available at https://github.com/insect-biome-atlas/utils-clean_asv_data.
<strong>Available data</strong>
The file 'shasum.txt' contains checksums for available files. Run 'shasum -c shasum.txt' to check file integrity.
Processed ASV data files
ASV taxonomic assignments, non-chimeric ASV cluster designations, consensus taxonomies, sequences of cluster representatives and summed counts of clusters in the sequenced samples are provided in compressed tab-separated files. Files are organized by country (Sweden and Madagascar), marked by the suffixes SE and MG, respectively.
Taxonomic assignments
The files asv_taxonomy_[SE|MG].tsv are tab-separated files with taxonomic assignments for all ASVs. Columns:

 ASV: The id of the ASV
 Kingdom, Phylum, Class, Order, Family, Genus, Species, BOLD_bin: Taxonomic assignment for each rank.

If an ASV was unclassified at a particular rank, the taxonomic label is prefixed with ‘unclassified.’ followed by the taxonomic assignment of the most resolved parent rank.
Cluster assignments
The files cluster_taxonomy_[SE|MG].tsv are tab-separated files containing all non-chimeric ASVs (that is, the ASVs passing the chimera-filtering step) with their corresponding taxonomic and cluster assignments. Columns:

 ASV: ASV id
 cluster: name of designated cluster
 median: the median of normalized reads across all samples for each ASV
 Kingdom, Phylum, Class, Order, Family, Genus, Species, BOLD_bin: taxonomic assignment of each ASV
 representative: contains 1 if ASV is a representative of its cluster, otherwise 0

Cluster counts
The files cluster_counts_[SE|MG].tsv are tab-separated files with read counts of ASV clusters (rows) in samples (columns). Counts have been summed for all ASVs belonging to each cluster.
Sequences of cluster representatives
The files cluster_reps_[SE|MG].fasta are text files in FASTA format with representative sequences for each cluster. The fasta headers have the format “&gt;ASV_ID CLUSTER_NAME”.
Consensus taxonomy
The files cluster_consensus_taxonomy_[SE|MG].tsv are tab-separated files with consensus taxonomy of each generated ASV cluster. Columns are the same as in asv_taxonomy_[SE|MG].tsv.
Cleaned, noise-filtered data
The files cleaned_noise_filtered_cluster_taxonomy_[SE|MG].tsv and cleaned_noise_filtered_cluster_counts_[SE|MG].tsv are tab-separated files with same information as in the ‘cluster_taxonomy’ and ‘cluster_counts’ files, but only for ASVs in clusters passing the noise filtering and cleaning steps.
<strong>References:</strong>

 Iwaszkiewicz-Eggebrecht, E., Łukasik, P., Buczek, M., Deng, J., Hartop, E. A., Havnås, H., ... & Miraldo, A. (2023). FAVIS: Fast and versatile protocol for non-destructive metabarcoding of bulk insect samples. <em>PloS one</em>, <em>18</em>(7), e0286272.
 Miraldo, A., Iwaszkiewicz-Eggebrecht, E., Sundh, J., Manoharan, L., Granqvist, E., Andersson, A., Łukasik, P., Roslin, T., Tack, A. J. M., & Ronquist, F. (2024). Amplicon sequence variants from the Insect Biome Atlas project (Version 1). SciLifeLab. https://doi.org/10.17044/scilifelab.25480681.v1
 Sundh, J. (2022). COI reference sequences from BOLD DB (Version 4). SciLifeLab. https://doi.org/10.17044/scilifelab.20514192.v4

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