Skip to content

Pipeline design

SSUextract detects candidate regions, extracts their sequences, selects a marker-specific database, and reports taxonomy.

SSUextract pipeline from Infernal detection and marker-specific BLAST through default BLAST taxonomy or optional tree-neighbor classification

Click the figure to open the full-size SVG.

Detection and extraction

Infernal supplies 1-based inclusive coordinates. The extraction stage retains both endpoints and reverse-complements negative-strand intervals. The typed hit table carries sample, model, contig, coordinates, strand, and sequence identity into annotation.

RF00177 and RF01960 belong to Rfam clan CL00111 and can recognize the same SSU locus. For overlapping same-strand hits, SSUextract retains the lower Infernal E-value. Equal E-values are resolved by the higher bit score. An exact tie stops the run instead of selecting a model without supporting evidence.

Any overlapping pair of different models without an explicit scientific competition rule stops the run; SSUextract does not silently retain both hits when their relationship is unknown.

Marker-specific annotation

RF00177 sequences use the 16S rRNA gene BLAST index. RF01960 sequences use the 18S rRNA gene index. The selected curated or img profile supplies both indexes and their taxonomy tables.

Optional tree classification

--tree_classification adds one tree task per extracted gene. Both marker indexes are searched before alignment. The marker represented by most of the best 100 unique subjects is selected; best bit score and the accepted Infernal model resolve ties.

The selected 100 reference sequences and query are aligned with RF00177 or RF01960 using cmalign. Covariance-model insert columns are masked; match columns with more than 90% gaps are then removed. IQ-TREE 3 runs a fast search under GTR+F+R4 with 1,000 SH-aLRT replicates and one inference thread. SSUextract sorts references by patristic distance from the query and assigns the lowest common taxonomy of the five nearest named references. Equal-distance references at the fifth-neighbor boundary are included in the same assignment.

A selected marker with fewer than three reference subjects cannot yield a tree. That query retains its BLAST taxonomy and records the skipped tree attempt; other extracted genes continue through tree classification.

Results

The workflow writes extracted FASTA files, parsed hit tables, BLAST results, a per-hit summary, category counts, and Nextflow execution reports. Tree mode also writes reference sequences, alignments, trees, logs, QC, and a combined neighbor table. See output files for paths and contents.