55dfbb63-4304-4fa8-a7b6-17988e0b33df 1.0 PEMA: a Pipeline for Environmental DNA Metabarcoding Analysis 2020-03-12 2021-02-11 2021-02-10 PEMA is a HPC-centered, containerized assembly of key metabarcoding analysis tools. It supports the downstream analysis of four marker genes (16S/18S rRNA, ITS and COI) but also, by allowing the user to train the classifiers with custom reference databases, it can be used for further marker genes. By combining state-of-the art technologies and algorithms with an easy to get-set-use framework, PEMA allows researchers to tune thoroughly each study thanks to roll-back checkpoints and on-demand partial pipeline execution features. Haris Zafeiropoulos Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research haris-zaf@hcmr.gr https://metadatacatalogue.lifewatch.eu/geonetwork/srv/api/records/55dfbb63-4304-4fa8-a7b6-17988e0b33df/attachments/remote%20sensing.jpg remote sensing.jpg metagenomics remote sensing modelling e-DNA Metabarcoding 16S 18S ITS COI Marker gene analysis Taxonomy assignment GNU GPLv3 Sequence pre-processing https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ FASTQC is used to obtain an overall read-quality summary. Sequence pre-processing Sequence pre-processing http://www.usadellab.org/cms/?page=trimmomatic Trimmomatic is used for trimming steps. Sequence pre-processing Sequence pre-processing https://cutadapt.readthedocs.io/en/stable/ Cutadapt is used for ITS to address the variability in length of this marker gene. Sequence pre-processing Sequence pre-processing http://cab.spbu.ru/software/spades/ BayesHammer is taken from the SPAdes assembly toolkit to revise incorrectly-called bases. Sequence pre-processing Sequence pre-processing https://github.com/neufeld/pandaseq PANDAseq assembles the overlapping paired-end reads. Sequence pre-processing Sequence pre-processing https://pythonhosted.org/OBITools/welcome.html The “obiuniq” program of OBITools groups the identical sequences in every sample, keeping track of their abundances. Sequence pre-processing Sequence pre-processing https://github.com/torognes/vsearch/releases/tag/v2.9.1 The VSEARCH package is invoked for chimera removal. Sequence pre-processing OTU clustering https://github.com/torognes/vsearch/releases/tag/v2.9.1 VSEARCH is used for OTU clustering. OTU clustering OTU clustering https://github.com/tingchenlab/CROP In case of COI marker genes COI, an unsupervised probabilistic Bayesian clustering algorithm (CROP) can be selected to perform the OTU clustering step. OTU clustering ASVs inference https://github.com/torognes/swarm For all marker genes supported, PEMA invokes the Swarm V2 algorithm to infer ASVs. ASVs inference Taxonomy assignment https://github.com/lanzen/CREST For the 16S/18S rRNA and ITS marker genes, the LCAClassifier algorithm of the CREST set of resources and tools is used together with the Silva and the Unite database. Two versions of Silva are included in PEMA: 128 and 132. Phylogeny-based assignment is also available for 16S rRNA marker gene data using a custom reference tree of 1,000 Silva-derived consensus sequences. Taxonomy assignment of the OTUs or ASVs returned in OTU clustering / ASVs inference step Taxonomy assignment https://github.com/rdpstaff/classifier For the COI marker gene, PEMA supports the RDPClassifier and the Midori and Midori2 reference databases to assign taxonomy of the MOTUs. Taxonomy assignment of the OTUs or ASVs returned in OTU clustering / ASVs inference step Ecological downstream analysis https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0061217; http://joey711.github.io/phyloseq/index.html; https://cran.r-project.org/web/packages/vegan/index.html The phyloseq R package can be used for downstream ecological analysis of the taxonomically assigned OTUs or ASVs. This includes α- and β-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations between samples. Ecological downstream analysis of the taxonomic tables Alignment tool https://mafft.cbrc.jp/alignment/software/ For the alignment of the consensus sequences returned by the phat algorithm for building the reference tree. It is used in the PEMA framework every time a user asks for a phylogenetic tree with the OTUs/ASVs found. Alignment of consensus sequences Alignment tool https://cme.h-its.org/exelixis/web/software/papara/index.html Alignment of short reads to reference phylogenies and alignments. In PEMA it aligns the OTUs/ASVs using the alignment of the sequences used for the reference tree as a core to align to. Alignment of short reads Build the reference tree https://github.com/amkozlov/raxml-ng Build the reference tree, and as with MAFFT it is used to build a phylogeny tree based on the OTUs/ASVs retrieved if the user asks. Build the reference tree Sequence placement on a phylogenetic tree https://github.com/Pbdas/epa-ng Performs maximum likelihood-based phylogenetic placement of genetic sequences on a user-supplied reference tree and alignment. In the PEMA framework it is used to assign OTUs/ASVs retrieved by PEMA to the reference tree. Maximum likelihood-based placement of genetic sequences on a reference tree PhAT algorithm https://github.com/lczech/gappa/wiki/Subcommand:-phat Generates consensus sequences from a sequence database according to the PhAT method using the "gappa" package. Consensus sequences generator Workflow Studio https://www.arb-silva.de/no_cache/download/archive/current/Exports/ http://reference-midori.info/index.html https://unite.ut.ee data analysis data processing https://doi.org/10.1093/gigascience/giaa022 WWW:LINK-1.0-http--related Paper describing the code http://pema.hcmr.gr/ WWW:LINK-1.0-http--link Home page/Github https://hub.docker.com/r/hariszaf/pema WWW:LINK-1.0-http--link Docker Hub https://datasets.datalad.org/?dir=/shub/hariszaf/pema WWW:LINK-1.0-http--link Singularity Hub