Riboseq pipeline

Version Compatibility

Reference examples tested with: cutadapt 4.4+, umi_tools 1.1+, STAR 2.7.11+, bowtie2 2.5.3+, plastid 0.6+, riboWaltz 2.0+, RiboCode 1.2+, riborex 2.4+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags
• R: packageVersion('<pkg>') then ?function_name to verify parameters
• Python: pip show <package> then help(module.function) to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Ribo-seq Pipeline

"Analyze my ribosome profiling data from FASTQ to translation efficiency" -> Orchestrate UMI handling, trimming, rRNA depletion, footprint-aware alignment, periodicity QC, P-site calibration, ORF detection, and differential translation, gating each downstream analysis on library quality.

This is a workflow skill: it owns the chaining decisions and hand-offs, not the internals of any one step.

The governing principle

A Ribo-seq analysis is decided at four seams, two of them set at the bench before any sequencing.

1. The harvest method is the deepest commitment and it fixes which analyses are even valid. Cycloheximide (CHX) pre-treatment freezes elongating ribosomes but distorts codon-level dwell times (it can flip the codon-occupancy/tRNA-abundance correlation, Hussmann 2015); dwell-time / stalling / pausing analyses are only valid on flash-frozen, no-drug libraries. Gene-level footprint counts and TE are robust to CHX; codon-resolution analyses are not.
2. Deduplicate ONLY when a UMI is present. Ribosome footprints are ~28-30 nt and massively over-represented at abundant transcripts, so identical reads are mostly real signal; position-deduplicating a non-UMI library destroys it. With UMIs, dedup BOTH the genome and the transcriptome BAM.
3. 3-nt periodicity is a HARD gate, not a QC nicety. Frame-based ORF calling and P-site analyses require strong frame-0 periodicity; a library that lacks it supports only gene-level counts. Certify it (riboWaltz frame-0 fraction) BEFORE any ORF/stalling step.
4. P-site offsets are per-read-length, and TE is a count model. Never hardcode a single 28-nt offset or the read 5' end; calibrate per length. Differential translation efficiency is a count-based GLM (riborex/Xtail/anota2seq) on CDS counts from BOTH assays, never a ratio of ribo/RNA ratios.

Pipeline overview

FASTQ -> UMI extract -> trim -> rRNA remove -> STAR (EndToEnd) -> dedup (UMI only)
      -> periodicity QC (HARD GATE) + per-length P-site offsets -> [ORF detection | translation efficiency | stalling]

Step 1: Preprocess

Goal: Produce a clean, footprint-aware alignment.

Approach: Extract UMIs first (if present), trim with a permissive floor, deplete rRNA before alignment, align end-to-end, and deduplicate only with UMIs. See riboseq-preprocessing for the decision tables.

# UMI-extract (if present) -> trim -> rRNA remove -> STAR EndToEnd -> dedup (UMI only)
cutadapt -a CTGTAGGCACCATCAAT --discard-untrimmed -m 15 -M 40 -o trimmed.fq.gz reads.fq.gz
bowtie2 -x contaminant_index -U trimmed.fq.gz --un-gz noncontam.fq.gz -S /dev/null -p 8
STAR --genomeDir STAR_index --readFilesIn noncontam.fq.gz --readFilesCommand zcat \
    --alignEndsType EndToEnd --seedSearchStartLmax 15 --outFilterMismatchNmax 2 \
    --quantMode TranscriptomeSAM --outSAMtype BAM SortedByCoordinate --outFileNamePrefix ribo_
samtools index ribo_Aligned.sortedByCoord.out.bam

--quantMode TranscriptomeSAM writes a SEPARATE ribo_Aligned.toTranscriptome.out.bam alongside the sorted genome BAM. RiboCode and riboWaltz transcriptome paths consume the TRANSCRIPTOME BAM; the sorted genome BAM is for plastid/genome-coordinate steps. With UMIs, deduplicate the transcriptome BAM too (coordinate-sort it first, then plain umi_tools dedup --method directional; see riboseq-preprocessing), or its ORF/periodicity inputs stay PCR-inflated. Do NOT add --per-contig/--per-gene here: they treat all reads on a transcript as one position, collapsing the per-codon footprints periodicity depends on.

Step 2: Periodicity QC and P-site offsets

Goal: Certify the library and obtain per-length P-site offsets.

Approach: Run riboWaltz to filter periodic read lengths and calibrate offsets; the frame-0 fraction is the pass/fail metric. See ribosome-periodicity.

library(riboWaltz)
annotation <- create_annotation("annotation.gtf")
reads <- bamtolist("bams", annotation = annotation)
reads <- length_filter(reads, length_filter_mode = "periodicity", periodicity_threshold = 50)
offsets <- psite(reads, extremity = "auto")   # per-length P-site offsets

Either riboWaltz (above) or the plastid metagene generate + psite CLI (used in the example script) is acceptable for offsets; pick one per project.

Step 3: Detect ORFs

Goal: Call translated ORFs once offsets are known.

Approach: Run RiboCode; read lengths come from the metaplots config, and -l is the longest-ORF toggle. See orf-detection.

prepare_transcripts -g annotation.gtf -f genome.fa -o annot
metaplots -a annot -r ribo_Aligned.toTranscriptome.out.bam -o metaplots
RiboCode -a annot -c metaplots_pre_config.txt -A CTG,GTG -l no -p 0.05 -o ribocode_result

Step 4: Translation efficiency

Goal: Test differential translation with matched RNA-seq.

Approach: Count both assays over the CDS and use a count-based GLM; use anota2seq when buffering vs control matters. See translation-efficiency.

library(riborex)
res <- riborex(rnaCntTable = rna_cds_counts, riboCntTable = ribo_cds_counts,
               rnaCond = cond, riboCond = cond, engine = "DESeq2")
sig <- res[which(res$padj < 0.05), ]

Step 5: Optional analyses

Stalling/pausing (only on flash-frozen no-drug data; see ribosome-stalling) and initiation-site mapping (needs a harringtonine/LTM library; see initiation-site-mapping) run off the same aligned BAM and calibrated offsets.

Common Errors

Symptom	Cause	Fix
Downstream analyses all noisy	Periodicity QC skipped	Gate ORF/stalling on the frame-0 fraction first
P-site offsets look wrong	Single hardcoded offset across lengths	Calibrate per length with riboWaltz
RiboCode uses wrong read lengths	-l passed read lengths	Read lengths come from metaplots; -l is a toggle
TE hits dominated by low-count genes	Ratio testing	Use riborex/Xtail/anota2seq count GLMs

Related Skills

• ribo-seq/riboseq-preprocessing - UMI handling, trimming, rRNA removal, alignment
• ribo-seq/ribosome-periodicity - Periodicity QC and P-site calibration
• ribo-seq/orf-detection - Translated ORF calling
• ribo-seq/translation-efficiency - Differential TE and buffering
• ribo-seq/initiation-site-mapping - Start-codon mapping from TI-seq
• differential-expression/deseq2-basics - Count-based differential testing

References

• McGlincy NJ, Ingolia NT. 2017. Transcriptome-wide measurement of translation by ribosome profiling. Methods 126:112-129. doi:10.1016/j.ymeth.2017.05.028
• Lauria F, Tebaldi T, Bernabò P, Groen EJN, Gillingwater TH, Viero G. 2018. riboWaltz: Optimization of ribosome P-site positioning in ribosome profiling data. PLoS Comput Biol 14(8):e1006169. doi:10.1371/journal.pcbi.1006169
• Xiao Z, Huang R, Xing X, Chen Y, Deng H, Yang X. 2018. De novo annotation and characterization of the translatome with ribosome profiling data. Nucleic Acids Res 46(10):e61. doi:10.1093/nar/gky179
• Hussmann JA, Patchett S, Johnson A, Sawyer S, Press WH. 2015. Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast. PLoS Genet 11(12):e1005732. doi:10.1371/journal.pgen.1005732 (cycloheximide distorts codon-level dwell times)