Interview-Driven Workflow Construction and Repair in Galaxy

Free-form source summary — Santana et al. 2023, Science: Scf1, a Candida auris-specific adhesin

Source

Workflow intent

The paper is a wet-lab genetics/cell-biology study. Its computational content is two small, self-contained, short-read Illumina analyses, both of which the authors state were run on the Galaxy public server at usegalaxy.org. That makes this paper an unusually direct Galaxy-workflow reconstruction target: the tool chain named in the Methods is already a Galaxy tool chain.

The two analyses are independent and have different shapes:

Pipeline A is the better primary target (standard Galaxy tools end to end, 6 public paired-end runs, two clean contrasts). Pipeline B is fully specified in the Methods but has one non-Galaxy step (see Open questions).

Pipeline A — RNA-seq differential expression

Steps, tools, parameters (all as stated in the supplement)

# Step Tool named in paper Parameters stated
1 Read QC FastQC none stated (“read quality was assessed”)
2 Quality trimming Cutadapt “Phred cutoff score of 20”; no adapter sequence given
3 Splice-aware alignment RNA STAR “default parameters”
4 Feature quantification featureCounts none stated
5 Differential expression DESeq2 none stated
6 Significance filter (DESeq2 output filter) |fold change| > 2 and adjusted p-value < 0.05

Platform: “Galaxy web platform public server at usegalaxy.org”. No tool versions are given for any of the six steps.

Library / sequencing facts

Reference data

Sample data (public, resolvable — strong test-data leads)

BioProject PRJNA904261 (SRA study SRP409192), RNA-Seq, TRANSCRIPTOMIC, RANDOM selection, PAIRED, Illumina NextSeq 2000, submitted by University of Michigan. Six runs, 2 biological replicates × 3 conditions, ~22–30 M spots each (~2.2–3.0 Gbp per run):

Run Experiment Sample Condition
SRR22376032 SRX18346538 AR0382_A parent AR0382 (clade I, high adhesion), rep A
SRR22376031 SRX18346539 AR0382_B parent AR0382, rep B
SRR22376030 SRX18346540 AR0387_A AR0387 (clade I, low adhesion), rep A
SRR22376029 SRX18346541 AR0387_B AR0387, rep B
SRR22376028 SRX18346542 AR0382_tnSWI1_A AR0382 tnSWI1 (B9J08_003460) insertional mutant, rep A
SRR22376027 SRX18346543 AR0382_tnSWI1_B AR0382 tnSWI1, rep B

Taxon 498019 (Candidozyma auris, formerly Candida auris). BioSamples SAMN31835905–SAMN31835910.

Contrasts and expected biological result (useful as workflow assertions)

Pipeline B — AtMT T-DNA insertion-site mapping (WGS)

Steps, tools, parameters

# Step Tool named in paper Parameters stated
1 Read QC FastQC none stated
2 Quality trimming Trimmomatic “Phred quality cutoff of 20”; trimming mode (SLIDINGWINDOW / TRAILING / LEADING) not stated
3 Align to T-DNA plasmid BWA-MEM reference = linearized pTO128 (pPZP-NAT); minimum seed length = 50, band width = 2
4 Extract soft-clipped flanks extractSoftClipped from SE-MEI (https://github.com/dpryan79/SE-MEI) none stated; operates on the aligned BAM
5 Align flanks to host genome BWA-MEM default configuration; reference = C. auris B8441, GCA_002759435.2
6 Confirmation Sanger sequencing of each locus wet-lab, out of workflow scope

Platform: also stated as run on usegalaxy.org (the Galaxy statement covers the sequencing-data analysis in this section).

The unusual parameters in step 3 (seed 50, band width 2) are deliberate: they force long exact matches to the T-DNA and suppress gapped extension, so the read portion that is host genomic DNA stays soft-clipped rather than being forced into the plasmid alignment. Any reimplementation must preserve them.

Library / sequencing facts

Sample data

BioProject PRJNA904262 (SRA study SRP409193), WGS, GENOMIC, RANDOM, PAIRED, Illumina NextSeq 550. Two runs — these are pools, not single mutants:

Run Experiment Sample Spots Avg length
SRR22376034 SRX18346544 Pool_A 16,373,182 294 (2 × ~147)
SRR22376033 SRX18346545 Pool_B 19,413,010 293 (2 × ~147)

BioSamples SAMN31836173–SAMN31836174.

Reference data

Other computational / analytical methods in the paper (not sequencing workflows)

Recorded for completeness; none of these are plausible Galaxy workflow steps, and none are the workflow this pipeline should build.

Strains and other identifiers worth carrying forward

Assumptions carried forward

  1. The Galaxy-relevant workflow is Pipeline A (RNA-seq DE), with Pipeline B as a second candidate. The paper contains no other analysis that consumes sequencing reads. Everything else is wet-lab or desktop software.
  2. The DESeq2 design is a simple one-factor, three-level condition model (AR0382 / AR0387 / tnSWI1) with n = 2, yielding two pairwise contrasts against AR0382. The paper states the contrasts and the significance thresholds but never writes out a design formula; n = 2 is inferred from the six deposited runs, not stated in the Methods.
  3. Strandedness: the Ribo-Zero Plus Stranded Total RNA kit is reverse-stranded (dUTP) in Illumina’s standard chemistry, so featureCounts would run reverse-stranded. The paper does not say this; it is inferred from the kit name.
  4. The B8441 reference is used for RNA-seq alignment even for AR0382/AR0387 samples, i.e. reads from a different clade-I isolate are mapped to the B8441 assembly. This is what the Methods say; it is not a transcription error.
  5. “Cutadapt with a Phred cutoff score of 20” is read as quality trimming (-q 20), not adapter trimming — no adapter sequence is given anywhere.
  6. Tool versions for the Galaxy steps are unknowable from the paper; any Galaxy workflow built from this must pin its own versions and cannot claim to reproduce the authors’ exact versions.

Open questions

  1. featureCounts annotation is unspecified. No GTF/GFF source is named for the B8441 assembly. A workflow must choose one (NCBI RefSeq GFF for GCA_002759435.2, or FungiDB’s B8441 GFF) and that choice will shift gene IDs and counts. This is the single largest gap for reproducing Pipeline A.
  2. No tool versions for any Galaxy step (FastQC, Cutadapt, RNA STAR, featureCounts, DESeq2, Trimmomatic, BWA-MEM). Only non-Galaxy software is versioned (R 4.0.3, DescTools 0.99.49, survminer 0.4.9, Fiji 1.52, CellProfiler 3.1.9, FlowJo 10.8.2, Gen5 3.12).
  3. Trimmomatic trimming mode is unstated for Pipeline B — “Phred quality cutoff of 20” could be SLIDINGWINDOW, TRAILING, or AVGQUAL. Minimum-length filtering is likewise unstated for both pipelines.
  4. extractSoftClipped (SE-MEI) has no known Galaxy Tool Shed wrapper. Pipeline B cannot be assembled from stock Galaxy tools as written. Either a wrapper must be authored, or the step must be substituted (e.g. samtools/awk soft-clip extraction, or a clip-aware structural-variant caller) — a substitution changes the method and should be flagged as such, not presented as a faithful port.
  5. The pTO128 (pPZP-NAT) plasmid sequence has no public accession in this paper. Pipeline B’s step-3 reference is unresolvable from the publication alone; it would have to come from the cited AtMT method paper (ref. 52), from Addgene, or by request. This is a hard test-data blocker for Pipeline B.
  6. PRJNA904262 contains two pooled samples, not per-mutant runs. The Methods describe insertion-site identification “from mutants of interest”, but the deposited data are Pool_A / Pool_B. How pools map to the individual mutants (tnSWI1, tnBCY1) — barcoding, demultiplexing, or post-hoc assignment — is not described. Any per-mutant claim from this data needs a step the paper does not document.
  7. No tnBCY1 RNA-seq runs are public even though Fig. S2 reports its transcriptome. A workflow test that tries to reproduce Fig. S2 has no input data.
  8. Fig. 2D’s cross-isolate SCF1 expression (r = 0.87, p = 8.4 × 10⁻⁸, 23 isolates) cannot come from RNA-seq — only 3 conditions were sequenced. It is almost certainly the RT-qPCR assay (ACT1-normalized, primers oTO1251/oTO1252), but the supplement does not explicitly tie the figure to the method. Not a sequencing-workflow concern, but do not model it as one.
  9. No variant-calling pipeline is described for the “206 coding SNPs between AR0382 and AR0387” claim or for Table S1’s allelic comparison. If a downstream design brief wants comparative genomics, it is inventing a step this paper does not contain.
  10. Access note: the Methods exist only in the supplementary PDF. The PMC full-text XML (retrievable via NCBI eutils efetch db=pmc) carries the main text but stops at “Supplementary Material”; the publisher page (science.org) returns 403 and the article is not in the Europe PMC open-access set. Anyone re-deriving this summary must fetch NIHMS2004453-supplement-2.pdf specifically.