rnastructurome

Allow dovetailing mates (--dovetail).

Bowtie2 --dpad padding around DP table.

Bowtie2 --ma match bonus used with --local.

Bowtie2 --mp mismatch penalties as max,min.

Bowtie2 alignment preset. Default is equivalent to --local -N 0 -D 20 -R 3 -L 20 -i S,1,0.50.

Bowtie2 --rdg read gap penalties as open,extend.

Bowtie2 --rfg reference gap penalties as open,extend.

Enable Bowtie2 local alignment mode (--local).

Report all valid alignments (-a).

Bowtie v1 --chunkmbs memory chunk size.

Optional path to a prebuilt Bowtie index (currently not consumed by workflow logic).

Report up to this number of alignments per read (-k).

Bowtie v1 -m maximum reportable alignments per read before suppression.

Bowtie v1 -n seed mismatch mode value.

Trim this many bases from 3' read end (--trim3).

Trim this many bases from 5' read end (--trim5).

Bowtie v1 -v mismatch mode value.

Genome route (STAR) quantification method. Default (false) uses STAR --quantMode TranscriptomeSAM projected onto transcripts, tag-corrected with samtools calmd, counted with rf-count. Set true for the legacy rf-count-genome + rf-rctools extract path.

Run SAMtools markdup deduplication on non-UMI libraries. Off by default: position-based duplicate removal is not valid for chemical-probing libraries without UMIs, where reads sharing a 5' start are independent RT events rather than PCR duplicates. UMI libraries are always deduplicated via UMI-tools.

STAR --sjdbOverhang for MaP alignment. Ideally readLength - 1; default 200 suits 201 bp reads.

Maximum number of loci a read is allowed to map to in STAR (--outFilterMultimapNmax). Reads mapping to more loci than this are discarded. Increase when studying repetitive non-coding RNAs.

Align against a transcript-level FASTA using Bowtie (RT-stop) / Bowtie2 (MaP). Default (false) downloads a genome FASTA and aligns with STAR.

SLURM account / allocation passed via --account.

Extra sbatch flags appended verbatim to process.clusterOptions.

SLURM partition / queue name passed via --partition.

Display the help message.

Display the full detailed help message.

Custom MultiQC yaml file containing HTML including a methods description.

Display hidden parameters in the help message (only works when --help or --help_full are provided).

Email address for completion summary.

Path to comma-separated file containing information about the samples in the experiment.

MultiQC report title. Printed as page header, used for filename if not otherwise specified.

The output directory where the results will be saved. You have to use absolute paths to storage on Cloud infrastructure.

Base quality threshold for 3' trimming.

Base quality threshold for 5' trimming (used for MaP; RT-stop is forced to 0).

Global fallback 3' adapter sequence(s), comma-separated. If omitted and no per-sample adapter is provided, the pipeline defaults to AGATCGGAAGAGC.

Global fallback 5' adapter sequence(s), comma-separated. If omitted and no per-sample adapter is provided, the pipeline defaults to AGATCGGAAGAGC.

Discard reads in which no adapter was found. For short-insert libraries (e.g. tRNA, miRNA) where every genuine read runs into the adapter.

Minimum read length retained after trimming.

Minimum adapter overlap required for trimming.

Enable cutadapt --nextseq-trim with this 3' quality cutoff, trimming high-quality poly-G tails left by 2-colour (NextSeq/NovaSeq) chemistry. Off by default.

If true, skip adapter trimming and apply quality/length trimming only.

Trim terminal N bases.

Ensembl FTP base URL used when auto-resolving transcript FASTA.

Ensembl release for transcript FASTA auto-resolution (current, latest, or release number like 114).

Path to transcript FASTA file.

Path to transcript annotation GTF file.

rf-correlate ignores sequence differences (e.g. SNVs) between compared transcripts (-I).

Generate the rf-correlate correlation heatmap PDF (-g; requires R).

rf-correlate minimum number of values to calculate a correlation (-m); a value between 0 and 1 is interpreted as a fraction of transcript length. Unset uses the tool default (off).

Run rf-correlate to compute pairwise reactivity-profile correlations between replicates of each sample group (a replicate-reproducibility QC surfaced in MultiQC). Only runs for sample groups with more than one replicate; a no-op otherwise.

Four-base DMS analysis (Mitchell et al., 2023) for DMS-MaP samples at pH >= 8: rf-count keeps only G->C/G->T mutations on G and drops indels (-om H2N,G2Y -ncl), and rf-norm normalises each base separately over the whole transcript (-4d).

Path to a reference .db structure file for rf-jackknife normalisation assessment. When provided, rf-jackknife runs between rf-norm and rf-fold and the output CSV reports optimal slope/intercept values. When omitted, rf-fold runs directly using whatever slope/intercept params are set.

Generate template-based 2D RNA structure diagrams using R2DT, coloured by normalised SHAPE/chemical-probing reactivity.

Comma-separated GTF biotypes sent to R2DT. R2DT only has templates for structured ncRNA classes; transcripts of other biotypes (mRNA, lncRNA, ...) are excluded so R2DT never force-fits them to the wrong template, and are drawn by ViennaRNA instead. Matched case-insensitively against transcript/gene biotype.

Cap reactivities to this value before Pearson correlation in rf-correlate (--cap-react). Not applied to the Spearman run, which is rank-based.

Discard reads with excessive 5' or 3' end clipping in rf-count / rf-count-genome (--discard-clipped-reads).

Block size for per-chromosome memory allocation in rf-count-genome (-bs); increase for large genomes.

Minimum MAPQ score for rf-count-genome (--map-quality). Useful for filtering multi-mappers; 0 keeps all alignments.

SAMtools working threads per processor instance in rf-count-genome (-wt); total threads = -p × -wt.

Deprecated: rf-count plot generation (-g) is always enabled.

In mutation mode (-m), collapse consecutive mutations/indels (--collapse-consecutive). Auto: on unless RT_enzyme is a Group II Intron RT (e.g. TGIRT); leave unset to use the auto default.

MaP only: discard mutations within N nt of each other (-dc N). Auto: 3 when RT_enzyme is a Group II Intron RT (e.g. TGIRT), off for M-MLV; leave unset to use the auto default.

In mutation mode (-m), discard reads shorter than this value (--discard-shorter).

MaP only: also evaluate quality of ±1 nt surrounding each mutation (-es).

In mutation mode with collapsing enabled, max distance to collapse (--max-collapse-distance).

In mutation mode (-m), downsample each transcript above this coverage in rf-count (--max-coverage). Unset means off. Ignored with --count_genome true.

MaP only: discard reads with median Phred+33 base quality below this value (-eq).

In mutation mode (-m), minimum Phred+33 base quality for mutations (--min-quality).

MaP only: ignore ambiguously aligned deletion events (-na). Auto: off for M-MLV, on for Group II Intron (e.g. TGIRT); leave unset to use the auto default.

MaP only: ignore all deletion events during mutation counting (-nd).

MaP only: ignore insertion events during mutation counting (-ni). Auto: off for M-MLV, on for Group II Intron (e.g. TGIRT); leave unset to use the auto default.

MaP only: mark only the right-most base of a deletion as mutated (--right-deletion). Auto: on for M-MLV, off for Group II Intron (e.g. TGIRT); leave unset to use the auto default.

In mutation mode (-m), pre-sort paired-end reads by read name (--sort-by-read-name). No effect on single-end data.

Path to rf-count mask file (--mask-file).

For paired-end samples, use only reads with both mates mapped (--paired-only).

Use only primary alignments in rf-count (--primary-only).

For paired-end samples, use only properly paired reads (--properly-paired).

Library strandedness fallback for rf-count-genome when RSeQC inference is unavailable (e.g. viral/bacterial references without a usable BED annotation).

Trim this many bases from the 5' read end for rf-count (-t5).

Exclude terminal base-pairs from rf-eval calculations (-it).

Generate R metric plots in rf-eval (-g).

Retain lonely/isolated base-pairs in rf-eval (-kl).

Retain pseudoknotted base-pairs in rf-eval (-kp).

Cutoff for classifying a base as highly-reactive in rf-eval unpaired-coefficient calculation (-c).

Path to a .db file of known RNA secondary structures. Enables rf-eval when provided.

Treat terminal base-pairs as unpaired in rf-eval (-tu).

Optional manifest of sub-region references, one per line: ref_seq_id start end structure_id [strand] (1-based inclusive, in the XML's own coordinate space). When set, rf-norm reactivities are sliced to each window before rf-eval, so a sub-region structure (e.g. an Rfam element on a whole chromosome) is scored against a matching windowed XML instead of the diluted full transcript. structure_id must match an entry in --rfeval_reference.

Write CT format structures with rf-fold (-ct). Disabled by default to keep dot-bracket output.

Generate dot plots from rf-fold (-dp).

Constraint file for allowed base-pairing positions (-c).

Generate ViennaRNA RNAplot structure diagrams via rf-fold (-g). Disabled by default; slow on large transcriptomes. Use --r2dt for template-based diagrams instead.

Intercept for reactivity-to-folding-constraint conversion in rf-fold (-in). Auto by chemical: DMS -2, NAI -0.8, 2A3 -0.4, other/unset -0.6. Overridden by jackknife calibration when available.

Only fold transcripts covered in at least this number of XML experiments (-oc).

Partition-function window size in rf-fold (-pw N).

Compute and report Shannon entropy in rf-fold (-sh).

Slope for reactivity-to-folding-constraint conversion in rf-fold (-sl). Auto by chemical: DMS 4.6, NAI 2.2, 2A3 1, other/unset 1.8. Overridden by jackknife calibration when available.

Fold without reactivity constraints in rf-fold (-i).

Fold a group's replicates together with RNAalifold, constrained by all their reactivity profiles at once, instead of folding each with RNAfold and aggregating base pairs (-ua). Applies only to groups with more than one replicate.

Use ViennaRNA hard constraints with rf-fold (-hc).

Maximal base-pair span for ViennaRNA in rf-fold (-md).

Pass ViennaRNA no-lonely-pairs mode to rf-fold (-nlp).

Path to ViennaRNA RNAplot binary for the ViennaRNA and rf-structextract SVG structure plots.

Enable windowed MFE folding in rf-fold with this window size (-w -fw N). Set null/unset to fold the whole transcript instead.

Generate R heatmap of grid-search results in rf-jackknife (-g).

Comma-separated intercept range to search in rf-jackknife (-in), e.g. -3,0.

Step size for intercept grid search in rf-jackknife (-is).

Retain lonely base-pairs (1 bp helices) in the reference structure before comparison (-kl).

Retain pseudoknotted base-pairs in the reference structure during FMI comparison (-kp). Disabled by default because rf-fold cannot predict pseudoknots, so including them systematically lowers FMI.

Use modified FMI (Lan et al. 2022) instead of standard FMI in rf-jackknife (-m).

Only use transcripts present across all replicates in rf-jackknife (-oc).

Pool XMLs from all sample groups into a single rf-jackknife run (output: jackknife/all_groups/) instead of running one jackknife per group. Default: true.

Use relaxed FMI criteria (Deigan et al. 2009) in rf-jackknife (-x).

Additional rf-fold parameters passed inside rf-jackknife (-rp), e.g. -md 500.

Comma-separated slope range to search in rf-jackknife (-sl), e.g. 0,5.

Step size for slope grid search in rf-jackknife (-ss).

Enable dynamic normalization window (--dynamic-window). Set to a positive integer to enable (value is passed to --norm-window); set to false or 0 to explicitly disable even for DMS (which enables it by default). Leave unset to use the per-method default.

Set reactivities lower than untreated to zero for Ding or Siegfried scoring (--ignore-lower-than-untreated).

Deprecated: rf-norm plot generation (--img) is always enabled.

Discard transcripts with mean coverage below this threshold (--mean-coverage).

Discard transcripts with median coverage below this threshold (--median-coverage).

Positions with read coverage below this threshold are reported as NaN (--nan). Auto: 1000 for MaP, 50 for RT-stop. Set to 0 to disable NaN masking.

Normalize each reactive base independently (--norm-independent).

Override rf-norm normalization method (-nm). 1=2-8%, 2=90% Winsorizing, 3=Box-plot, 4=Mitchell (MaP only).

Normalization window size (--norm-window). Auto: 50 for DMS or RT-stop, unset otherwise; leave unset to use the auto default.

rf-normfactor minimum coverage (-mc): bases below this coverage are excluded from the normalization factor calculation. Auto: 1000 for MaP, 50 for RT-stop; leave unset to use the auto default.

--count_genome true only: after rf-rctools extract, keep transcripts with at least one position at this coverage or higher before rf-norm. Set to 0 to keep the full annotation RC.

Pseudocount used by Ding scoring (--pseudocount).

Score raw reactivities without applying normalization (--raw).

Reactive bases used for normalization window selection (--reactive-bases), e.g. AC for DMS.

Remap normalized reactivities to the 0-1 range according to Zarringhalam et al. (--remap-reactivities).

Override rf-norm scoring method (-sm). 1=Ding, 2=Rouskin, 3=Siegfried, 4=Zubradt.

Cross-experiment normalization via rf-normfactor (one factor set per reference, fed to rf-norm via -nf), putting reactivities on a common scale. Leave unset for auto (enabled only for a reference with more than one treated sample to cross-normalise); set true to force on, false to force off (per-sample box-plot).

Normalization window offset (--window-offset).

GTF attribute used as the output RC entry ID by rf-rctools (-b).

GTF feature type to extract with rf-rctools (-f); override for non-standard annotations.

Restrict WIG output to specific bases using an IUPAC code or combination (e.g. AC for DMS probing) (-kb). Default: all bases (N).

Report positions with zero reactivity in the WIG/BigWig output (-z). Enabled by default so genome browsers receive complete tracks without gaps.

Path to R executable used by rf-count, rf-norm, and rf-fold plotting.

Stop the pipeline after rf-jackknife completes, skipping rf-fold, structure visualisation, browser track generation, and RDAT output. Useful for calibration runs where you only want jackknife statistics.

Run rf-structextract after rf-fold to extract high-confidence, low-reactivity / low-Shannon structural motifs from the folded structures.

rf-structextract preserves dinucleotide frequencies when shuffling for energy evaluation (-ds).

rf-structextract only reports elements with a free energy significantly lower than expected by chance (-ee).

rf-structextract skips low-reactivity evaluation (-ir). When false (default), reactivity is evaluated so only regions with probing support are extracted.

rf-structextract skips low-Shannon evaluation (-is). When false, the Shannon-entropy test is applied so only high-confidence regions are extracted.

rf-structextract discards elements with a loop larger than this (-xl). Unset for no limit.

rf-structextract discards structure elements longer than this (-xm). Unset for no limit.

rf-structextract minimum fraction of bases whose Shannon and reactivity are below the transcript median (-mb).

rf-structextract discards structure elements shorter than this (-mm).

rf-structextract discards elements with less than this fraction of paired bases (-mp).

rf-structextract skips low-reactivity/low-Shannon evaluation for transcripts below this length (-ml).

rf-structextract windows with less than this fraction of bases covered are set to NaN (-mv).

rf-structextract only reports elements encompassing multiway junctions (-mo).

rf-structextract number of sequence shufflings for energy evaluation (-ns).

rf-structextract reports each extracted element in a separate file (-opf).

Render an SVG diagram per extracted rf-structextract motif via ViennaRNA RNAplot.

rf-structextract p-value threshold for energy significance (-v); requires --structextract_eval_energy.

rf-structextract window size in nt for median reactivity/Shannon calculations (-w).

Fallback reverse transcriptase enzyme for rows missing RT_enzyme (e.g. M-MLV).

Fallback chemical probing reagent for rows missing chemical (e.g. 1M7).

Require an exact sample_group+replicate match when pairing treated samples with untreated/denatured controls. By default, a treated group with no exact match falls back to the control whose sample_group shares the longest leading substring with it (see the usage docs). With this set, unmatched treated groups proceed without an untreated control (scoring method 2 or 4).

Fallback probing method for rows missing method.

Fallback organism for rows missing organism, typically a Latin binomial such as Homo sapiens.

Fallback DMS reaction pH for rows missing pH.

Fallback probing principle for rows missing principle.

Fallback sample ID for rows missing sample_id.

Fallback UMI pattern for rows missing umi_pattern. Supplying a pattern enables umi_tools extract before cutadapt.