Automated pipeline for downloading extremophile sequencing data, classifying runs as WGS (shotgun) or 16S amplicon, and routing each to the appropriate assembly and annotation workflow.
This pipeline takes a TSV export from any of our extremophile databases (GlaciaBase, TardiBase, or ExtremoDex) and processes the sequencing runs through a fully automated workflow. The critical first step is classifying each run as WGS (whole-genome shotgun) or 16S amplicon, because these two data types require fundamentally different analysis approaches and produce different kinds of biological insight.
1. Data Download — Uses the SRA Toolkit (prefetch + fasterq-dump) to download
raw FASTQ files from NCBI for each SRR accession in your input file.
2. Classification — Reads the ASSAY_TYPE column from your export to route each run to the correct
branch. Runs labeled AMPLICON go to the 16S pipeline; runs labeled WGS go to the
shotgun metagenome assembly pipeline.
3. Quality Control — Both branches start with fastp for adapter trimming, quality
filtering, and per-base quality reporting.
4. Branch-specific analysis — WGS runs get assembled into contigs and binned into MAGs;
amplicon runs get denoised into ASVs and classified taxonomically.
Shared WGS only Amplicon only
The pipeline splits at the classification step. Each branch uses purpose-built tools for its data type.
This distinction is one of the most important decisions in metagenomics. The two approaches generate fundamentally different kinds of data, and using the wrong analysis on the wrong data type will produce misleading or meaningless results.
16S rRNA amplicon sequencing targets a single conserved gene (~1,500 bp) to identify which organisms are present in a community. It tells you who is there and in what proportions, but it gives you almost no information about the functional gene content of those organisms. You cannot do ortholog analysis, functional pathway reconstruction, or genome assembly from 16S data — there simply isn't enough genomic information in a single marker gene.
Whole-genome shotgun (WGS) metagenomics sequences random fragments from all genomic DNA in the sample. This gives you both taxonomic composition AND functional information — you can assemble genomes (MAGs), identify genes, reconstruct metabolic pathways, and perform ortholog analysis across species. WGS is the only approach that supports genome-level analysis.
| Feature | WGS Shotgun | 16S Amplicon |
|---|---|---|
| Target | All genomic DNA in sample | 16S rRNA gene only (~1.5 kb) |
| Taxonomic resolution | Species / strain level | Genus level (sometimes species) |
| Genome assembly | Yes — MAGs via binning | No — not enough info |
| Ortholog analysis | Yes | No — insufficient data |
| Functional pathways | Yes — gene prediction + annotation | No — only taxonomy |
| Cost per sample | Higher (deeper sequencing needed) | Lower (targeted amplification) |
| PCR bias | Minimal | Yes — primer bias |
| Detects viruses | Yes | No — no 16S in viruses |
| Eukaryote detection | Yes | Limited — 18S/ITS needed |
| Best for | Functional genomics, MAGs, comparative genomics | Community surveys, biodiversity, rapid profiling |
The pipeline reads the ASSAY_TYPE column from your database export. GlaciaBase, TardiBase,
and ExtremoDex all annotate runs with their assay type (AMPLICON or WGS) based on NCBI SRA metadata.
The pipeline uses this annotation to automatically route each run to the correct analysis branch —
no manual sorting required.
Runs classified as WGS get assembled with MEGAHIT, binned with MetaBAT2, quality-checked against the MIMAG standard (>90% complete, <5% contamination = high quality), and taxonomically classified with GTDB-Tk. Amplicon runs go through QIIME2/DADA2 for denoising into ASVs, SILVA for taxonomy, and standard alpha/beta diversity calculations.
Export data from any of these databases to generate the TSV input file for the pipeline. Each database's Extract Data tab lets you select runs and download a TSV with the required columns (SRR accession, BioProject, assay type, organism).
Psychrophile sequencing database. Cold-adapted organisms from polar, alpine, deep-sea, and permafrost environments. Contains WGS and amplicon data for metagenome communities and individual species.
Tardigrade genomics database. Sequencing runs for 39+ tardigrade species with cryptobiosis types, survival tolerances, and habitat data. Primarily WGS and RNA-Seq data.
The extremophile rolodex. Aggregates data across all extremophile categories — psychrophiles, thermophiles, halophiles, acidophiles, radioresistant organisms, and polyextremophiles.
The input is a tab-separated file with four columns. You can export this directly from any database's Extract Data tab, or create it manually: