Anacapa Toolkit: an environmental DNA toolkit for processing multilocus metabarcode datasets

Abstract

are co-equal second authors.Robert Wayne and Rachel S. Meyer are co-equal senior authors. Abstract 1. Environmental DNA (eDNA) metabarcoding is a promising method to monitor species and community diversity that is rapid, affordable and non-invasive. The longstanding needs of the eDNA community are modular informatics tools, comprehensive and customizable reference databases, flexibility across high-throughput sequencing platforms, fast multilocus metabarcode processing and accurate taxonomic assignment. Improvements in bioinformatics tools make addressing each of these demands within a single toolkit a reality.2. The new modular metabarcode sequence toolkit Anacapa (https ://github.com/ limey-bean/Anaca pa/) addresses the above needs, allowing users to build comprehensive reference databases and assign taxonomy to raw multilocus metabarcode sequence data. A novel aspect of Anacapa is its database building module, "Creating Reference libraries Using eXisting tools" (CRUX), which generates comprehensive reference databases for specific user-defined metabarcoding loci. The Quality Control and ASV Parsing module sorts and processes multiple metabarcoding loci and processes merged, unmerged and unpaired reads maximizing recovered diversity. DADA2 then detects amplicon sequence variants (ASVs) and the Anacapa Classifier module aligns these ASVs to CRUX-generated reference databases using Bowtie2. Lastly, taxonomy is assigned to ASVs with confidence scores using a Bayesian Lowest Common Ancestor (BLCA) method. The Anacapa Toolkit also includes an r package, ranacapa, for automated results exploration through standard biodiversity statistical analysis.3. Benchmarking tests verify that the Anacapa Toolkit effectively and efficiently generates comprehensive reference databases that capture taxonomic diversity, and can assign taxonomy to both MiSeq and HiSeq-length sequence data. We demonstrate the value of the Anacapa Toolkit in assigning taxonomy to seawater eDNA samples collected in southern California.

Beach environmental DNA fills gaps in photographic biomonitoring to track spatiotemporal community turnover across 82 phyla

“…Libraries for 88 sites and negative controls were sequenced in two runs: 3.34 Gb of fastq data were generated in the first run (170818_300PE_MS1) and 12.25 Gb were generated in the second run (171006_300PE_MS1)(NCBI SRA PENDING ACCEPTANCE). Results were output from the Anacapa Toolkit (Curd et al, 2019) summarized to the Least Common Ancestor, and then taxa were removed that were found in negative controls as well as taxa with only one read across all samples, yielding 1468 total taxa for CO1, 2689 for 18S, 2593 for 16S, and 43 for 12S (Tables S4-S7). The total unique taxa assigned to kingdoms across all eDNA results were 1132 Animalia, 27 Archaea, 2,533 Bacteria, 1,588 Chromista, 516 Fungi, 433 Plantae, and 154 Protozoa, with 3 unassigned to a kingdom.…”

“…The metabarcoding approach, which amplifies and sequences barcode loci from a mixed sample (Taberlet et al, 2012; 2018), seeks to match DNA reads to reference sequences from voucher specimens to receive a taxonomic assignment (see Cristescu, 2014 for discussion). DNA barcode reference databases still have large gaps across phylogenies (see Curd et al, 2019) that limit discovery of their true taxonomic membership. Moreover, many barcodes lack diagnostic sequence variants for lower taxonomic assignment (Wolfe et al, 1987).…”

“…Demultiplexed library Fastq files were processed with the Anacapa Toolkit (archived version (doi:10.5281/zenodo.3064152)(github.com/limey-bean/Anacapa; Curd et al, 2019) using default parameters. In brief, reads were processed first by cutadapt (Martin, 2011) to remove adapters and any 3’ primer reverse complement.…”

“…Reference databases for 16S, 18S, CO1, and 12S were generated with the CRUX step of the Anacapa Toolkit (Curd et al, 2019; DOI: https://doi.org/10.5061/dryad.mf0126f) that queried NCBI nr/nt databases. The CO1 database was subsequently modified by appending all BOLD CO1-5P sequences, accessed via the BOLD API on September 24, 2018 and comprising 5.08 million sequences, and also appending 847 recently generated invertebrate CO1-5P barcode sequences from the LACM DISCO program (subsequently published in BOLD with the same identifiers).…”

PUMAA: A Platform for Accessible Microbiome Analysis in the Undergraduate Classroom

“…Even with the increasing availability of GUI analysis tools, there is still the problem that the data output file formats from QIIME or custom commercial and academic pipelines such as MrDNA ( mrdnalab, 2020 ) and Anacapa ( Curd et al, 2019 ) do not match the data input file formats required for the GUI and web-based analysis and visualization tools. Formatting the different analysis pathway files into a single pipeline is a non-trivial task requiring either running scripts or hours of manual reformatting.…”