Version Compatibility

Reference examples tested with: cutadapt 4.4+, miRge3.0 0.1.4+, miRDeep2 2.0.1.3+, DESeq2 1.42+, apeglm 1.24+, miRanda 3.3a, umi_tools 1.1+, miRTrace 1.0+

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

• R: packageVersion('<pkg>') then ?function_name to verify parameters
• CLI: <tool> --version then <tool> --help to confirm flags

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

Note: the correct 3' adapter sequence and the UMI/4N scheme are KIT-specific (TruSeq vs NEXTflex vs QIAseq) — confirm against the kit before trimming. The normalizer that best fits compositional miRNA data drifts with the method literature; treat the table below as a decision aid to validate, not a fixed rule.

Small RNA-seq Pipeline

"Analyze my small RNA-seq data from FASTQ to differential miRNAs" -> Chain kit-aware trimming, known-miRNA quantification (or novel discovery), a compositionally-aware DE test, and expression-filtered target prediction.

• CLI + R: cutadapt -> (miRge3.0 | miRDeep2) -> DESeq2 (inspect size factors) -> miRanda (filter by anti-correlated mRNA)

This is a workflow skill: it owns the chaining decisions and hand-offs, not the internals of any one step. Every step below cross-references the component skill that teaches its mechanism.

The governing principle

A small RNA-seq result turns on two things the mRNA pipeline never faces — the adapter is on every read, and the counts are compositional — so the trustworthy result is decided at these seams.

1. The library kit fixes adapter + UMI/4N handling, committed once at trimming and un-reversible. The insert (~22 nt) is far shorter than the read, so the 3' adapter is on EVERY real read and --discard-untrimmed correctly drops no-insert junk (the inverse of genomic DNA). NEXTflex 4N random bases are stripped AFTER adapter removal; QIAseq UMIs are extracted and deduped. NEVER position-dedup a non-UMI small-RNA library — abundant miRNAs legitimately share start positions, so position-dedup destroys real signal.
2. The normalizer is the dominant analytic commitment — more than the DE model. miRNA counts are few-featured and compositional; a handful of miRNAs can dominate the library. Global scaling (TMM's 30%/5% trimming, median-of-ratios) is built for ~20k mRNAs and is harmful among hundreds of miRNAs (Garmire 2012). The normalizer choice CHANGES which miRNAs are called DE. Commit and report it; do not default silently.
3. RAW counts (not RPM) flow into DESeq2, and size factors must be inspected. A large rise in one abundant miRNA mechanically deflates all others (the closed-composition artifact), manufacturing spurious "down" calls. Confirm the input is raw integer counts and inspect sizeFactors(dds) before trusting any call.
4. Targets are hypotheses, not findings. miRanda output must be intersected with anti-correlated mRNA DE and validated databases before interpretation.

Pipeline map

FASTQ (single-end small RNA)
  | [1] Trim (kit-aware) --------> cutadapt (+ umi_tools / 4N)   (small-rna-seq/smrna-preprocessing)
  v     ^-- commitment: kit adapter + UMI/4N; --discard-untrimmed; NO position-dedup w/o UMI
  | [2] Quantify OR discover ----> miRge3.0 (known+isomiR) | miRDeep2 (novel)  (small-rna-seq/mirge3-analysis, mirdeep2-analysis)
  v
  | [3] Differential expression -> DESeq2 on RAW counts; INSPECT size factors   (small-rna-seq/differential-mirna)
  v     ^-- commitment: the NORMALIZER (drives which miRNAs are DE)
  | [4] Target prediction -------> miRanda, filtered by anti-correlated mRNA DE  (small-rna-seq/target-prediction)
  v
Differential miRNAs + expression-supported targets
   (tRF/piRNA reads -> small-rna-seq/trf-pirna-profiling)

Made-once commitments

| Commitment | Choice | Consequence inherited downstream | |------------|--------|----------------------------------| | Library kit adapter + UMI/4N | TruSeq / NEXTflex-4N / QIAseq-UMI handling, set at trimming | Wrong adapter or missed 4N/UMI corrupts every count; non-UMI position-dedup destroys real miRNAs | | Quantification target | Known-miRNA + isomiR (miRge3) vs novel discovery (miRDeep2) | Discovery is high-FP and needs a genome + bowtie1; quantification is the common curated case | | Normalizer | median-of-ratios/TMM vs quantile/loess vs RUVg vs CoDA/ALDEx2 | Which miRNAs are DE (more than the DE model choice) | | Biofluid vs tissue | plasma/serum has NO trustworthy endogenous normalizer | DE may be uninterpretable without spike-ins + hemolysis modeling |

The canonical order and why

1. Trim (kit-aware) — adapter removal with --discard-untrimmed; 4N/UMI handling before or during collapse. Order-trap: position-deduping a non-UMI library removes real high-abundance miRNAs.
2. miRTrace QC — length/complexity, RNA-class composition, cross-clade contamination BEFORE quantification (a good mapping rate hides sample swaps and reagent contamination).
3. Quantify (miRge3) or discover (miRDeep2) — to raw integer counts. For miRDeep2, choose the score cutoff from survey.pl signal-to-noise, not a fixed rule.
4. Low-count filter (rowSums >= 10, lower than mRNA) BEFORE normalization.
5. DE with the committed normalizer — raw counts into DESeq2; inspect sizeFactors. Order-trap: feeding RPM/CPM bakes compositional distortion into an invalid model.
6. Target prediction — intersect miRanda predictions with anti-correlated mRNA DE (order-trap: enrichment on raw predictions is false-positive-laden).

Fork/decision points

Pipeline-level selection only; mechanism lives in the component skills.

| Fork | Lean toward | Hand off to | |------|-------------|-------------| | Quantify vs discover | miRge3.0 (known + isomiR, curated, common) vs miRDeep2 (novel only; high FP, genome + bowtie1, survey.pl cutoff) | small-rna-seq/mirge3-analysis, small-rna-seq/mirdeep2-analysis | | Normalizer | median-of-ratios/TMM (risky under composition shift) vs quantile/loess (better on skewed miRNA) vs RUVg (control/empirical miRNAs) vs CoDA/ALDEx2 (compositional sensitivity check) | small-rna-seq/differential-mirna | | Biofluid/plasma | no trustworthy endogenous normalizer (miR-16 is hemolysis-sensitive, U6 degrades); cel-miR-39 spike controls EXTRACTION not biological scale; model batch/hemolysis explicitly | small-rna-seq/differential-mirna | | RNA class | miRNA vs tRF/piRNA (26-32 nt peak) -> route to trf-pirna-profiling (MINTmap/unitas) | small-rna-seq/trf-pirna-profiling |

Primary path: cutadapt -> miRge3.0 -> DESeq2 -> miRanda

Goal: turn kit-specific FASTQ into differential miRNAs with expression-supported targets.

Approach: trim to the kit, quantify known miRNAs/isomiRs, test RAW counts with size-factor inspection, then filter targets by anti-correlation. (The runnable script in examples/ shows the miRDeep2 novel-discovery route below; the miRge3.0 commands are shown here.)

# Kit-aware trim: adapter on EVERY read, so --discard-untrimmed drops no-insert junk (inverse of gDNA).
# NEXTflex 4N: cutadapt -u 4 -u -4 AFTER adapter. QIAseq UMIs: umi_tools. NEVER position-dedup non-UMI.
cutadapt -a TGGAATTCTCGGGTGCCAAGG --minimum-length 18 --maximum-length 30 --discard-untrimmed \
    -o trimmed.fastq.gz reads.fastq.gz

# Known-miRNA + isomiR quantification (the common case). NOTE: v3 has NO 'annotate' subcommand
# (that was miRge2); it is `miRge3.0 -s ...`. Reads are already cutadapt-trimmed, so -a is OMITTED
# (v3 has no 'none' keyword; passing one makes cutadapt treat it as an adapter sequence and fail).
# isomiRs are annotated by default; -gff emits the isomiR GFF.
# (-ai would instead compute A-to-I editing, a separate analysis, not isomiR reporting.)
miRge3.0 -s trimmed.fastq.gz -lib /path/to/miRge3_Lib -on human -db mirbase \
    -gff -cpu 8 -o mirge_out

library(DESeq2)
# RAW counts (miR.Counts.csv), NOT RPM. A few miRNAs can dominate, so inspect sizeFactors first.
# miRge3.0 writes into a timestamped subfolder (mirge_out/miRge.YYYY-M-D_h-m-s/), not directly into -o
counts <- read.csv(Sys.glob('mirge_out/miRge.*/miR.Counts.csv')[1], row.names = 1)
dds <- DESeqDataSetFromMatrix(round(counts), colData, ~condition)
dds <- dds[rowSums(counts(dds)) >= 10, ]      # lower prefilter than mRNA
dds <- DESeq(dds)
print(sizeFactors(dds))                       # a dominant-miRNA shift distorts these -> spurious calls
res <- lfcShrink(dds, coef = 'condition_treated_vs_control', type = 'apeglm')

# Targets are a hypothesis: intersect with anti-correlated mRNA DE before trusting them.
# -sc 140: miRanda default alignment-score floor; -en -20: keep duplexes with MFE <= -20 kcal/mol (stable binding)
miranda mature_mirnas.fa target_3utrs.fa -sc 140 -en -20 -strict -out targets.txt

Novel discovery alternative: miRDeep2

Goal: discover previously unannotated miRNAs (only when that is the question).

Approach: collapse reads, map to the genome with bowtie1, run miRDeep2, and pick the score cutoff from the signal-to-noise survey — not a fixed threshold. Full runnable script: examples/smrna_full_pipeline.sh.

gunzip -kc trimmed.fastq.gz > trimmed.fastq            # mapper.pl reads plain-text FASTQ, not gzip
mapper.pl trimmed.fastq -e -h -i -j -l 18 -m -p genome_index \
    -s reads_collapsed.fa -t reads_collapsed_vs_genome.arf
miRDeep2.pl reads_collapsed.fa genome.fa reads_collapsed_vs_genome.arf \
    mature_ref.fa none hairpin_ref.fa -t Human      # score cutoff from survey.pl signal-to-noise

QC checkpoints between steps

| After | Gate | Interpretation | |-------|------|----------------| | Trim | Read-length peak 21-23 nt | 30+ nt smear = degradation/tRNA/rRNA; 26-32 nt peak = piRNA (route to trf-pirna-profiling) | | miRTrace/align | RNA-class composition (miRNA vs tRF/rRF/piRNA); cross-clade contamination | Abundant non-miRNA classes mean this is a tRF/piRNA story, not a miRNA one | | Before DE | RAW counts (not RPM); size factors inspected | A dominant-miRNA shift distorts size factors and manufactures spurious "down" calls | | After DE | baseMean reported with every call | A significant fold-change on a ~5-count miRNA is noise | | Targets | filtered by anti-correlated mRNA DE + validated DBs | Raw miRanda predictions are hypotheses, not findings |

Ligation bias means absolute cross-miRNA abundance WITHIN a sample is untrustworthy; compare the same miRNA across samples, never across kits. For a fully reproducible run, nf-core/smrnaseq chains these steps (workflow-management/nf-core-pipelines).

Common Errors

| Symptom | Cause | Fix | |---------|-------|-----| | Real high-abundance miRNAs vanish | Position-deduped a non-UMI library | Dedup ONLY with UMIs (umi_tools); non-UMI libraries are not position-deduped | | Invalid model / distorted calls | Fed RPM/CPM to DESeq2 | Use RAW integer counts; RPM bakes in composition | | Many spurious "down" miRNAs | One abundant miRNA rose and deflated the rest (closed composition) | Inspect size factors; consider quantile/RUVg/ALDEx2 as a sensitivity check | | DE looks strong but is noise | Fold-change on a ~5-count miRNA | Report and gate on baseMean | | Plasma miRNA DE uninterpretable | No trustworthy endogenous normalizer; hemolysis confound | Spike-ins for extraction control + model hemolysis/batch explicitly | | "miRNA" library is mostly tRF/piRNA | 26-32 nt peak / broad tRF distribution | Route to trf-pirna-profiling (MINTmap/unitas) |

Pipeline map (hand-offs)

• small-rna-seq/smrna-preprocessing - kit-specific adapter, UMI, and 4N handling
• small-rna-seq/mirge3-analysis - known-miRNA and isomiR quantification
• small-rna-seq/mirdeep2-analysis - novel miRNA discovery and survey.pl cutoff
• small-rna-seq/differential-mirna - compositionally-aware DE and normalizer selection
• small-rna-seq/target-prediction - seed prediction filtered by expression
• small-rna-seq/trf-pirna-profiling - tRF and piRNA profiling

The runnable miRDeep2 discovery-route script is in this skill's examples/ (smrna_full_pipeline.sh).

Related Skills

• small-rna-seq/smrna-preprocessing - Kit-specific adapter, UMI, and 4N handling
• small-rna-seq/mirdeep2-analysis - Novel miRNA discovery
• small-rna-seq/mirge3-analysis - Known-miRNA and isomiR quantification
• small-rna-seq/differential-mirna - Compositionally-aware DE
• small-rna-seq/target-prediction - Seed prediction filtered by expression
• small-rna-seq/trf-pirna-profiling - tRF and piRNA profiling
• differential-expression/deseq2-basics - DESeq2 model, contrasts, shrinkage
• workflow-management/nf-core-pipelines - Run nf-core/smrnaseq as a curated, reproducible pipeline

References

• Garmire LX, Subramaniam S (2012) Evaluation of normalization methods in mammalian microRNA-Seq data. RNA 18:1279-1288. DOI 10.1261/rna.030916.111. (normalization, not the DE model, drives miRNA DE results.)
• Risso D, Ngai J, Speed TP, Dudoit S (2014) Normalization of RNA-seq data using factor analysis of control genes or samples. Nature Biotechnology 32:896-902. DOI 10.1038/nbt.2931. (RUVg for miRNA/unwanted variation.)
• Friedländer MR, Mackowiak SD, Li N, Chen W, Rajewsky N (2012) miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Research 40:37-52. DOI 10.1093/nar/gkr688.