Version Compatibility

Reference examples tested with: STAR 2.7.11+, samtools 1.19+, fastp 0.23+, deepTools 3.5+, PreSeq 3.2+, exomePeak2 1.14.x (Bioconductor 3.18 ONLY -- deprecated in Bioc 3.19, removed in 3.20; on current Bioc install from the Bioc 3.18 archive or use a successor), MACS3 3.0+, ChIPseeker 1.38+, Guitar 2.18+, BSgenome.Hsapiens.UCSC.hg38 1.4+, TxDb.Hsapiens.UCSC.hg38.knownGene 3.18+, HOMER 4.11+.

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.

exomePeak2 has NO mode= or experiment_design= argument; differential is triggered by populating bam_treated_ip + bam_treated_input. MeTPeak defaults are WINDOW_WIDTH=50, SLIDING_STEP=50, FRAGMENT_LENGTH=100. MACS3 default --keep-dup is 1 and MUST be overridden to all for non-UMI MeRIP. Guitar txTxdb= is the modern argument name (older releases used txdb=).

MeRIP-seq End-to-End Pipeline

"Analyze my MeRIP-seq data from FASTQ to differential m6A peaks" -> Orchestrate read alignment (STAR splice-aware to GENOME), IP-enrichment QC (deepTools plotFingerprint, replicate Spearman, PreSeq saturation), m6A peak calling (exomePeak2 transcript-aware default, MACS3 broad as cross-check), DRACH motif sanity check (HOMER), exomePeak2 differential via the four-BAM-vector interface, ChIPseeker feature annotation, and Guitar transcript-feature metagene confirming canonical stop-codon enrichment. Defer per-skill deep treatment to epitranscriptomics/merip-preprocessing, epitranscriptomics/m6a-peak-calling, epitranscriptomics/m6a-differential, and epitranscriptomics/modification-visualization.

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

The governing principle

MeRIP-seq inverts several DNA-pipeline reflexes; the trustworthy callset is decided at these seams.

1. Do NOT deduplicate non-UMI MeRIP — duplicates are signal, not artifact. A highly methylated, highly expressed transcript legitimately produces many identical fragments; removing them (or MACS3 default --keep-dup 1) erases the strongest m6A peaks. Keep all reads (--keep-dup all); only dedup when a UMI is present.
2. Enrichment is IP-vs-Input, and differential is a FOUR-BAM comparison. Every condition needs its own IP AND Input. exomePeak2 has no mode=/experiment_design= argument; differential is triggered simply by populating bam_treated_ip + bam_treated_input alongside the control bam_ip + bam_input.
3. DRACH is a peak-SET sanity check, never a per-peak filter. Confirm the motif is enriched across the whole peak set (P-value < 1e-50); post-hoc dropping individual peaks that lack a DRACH match discards real non-canonical sites and biases the callset.
4. The Guitar stop-codon metagene is the biological go/no-go. m6A concentrates near the stop codon / 3'UTR-proximal CDS end; if that enrichment is absent, the IP failed or the antibody is wrong — STOP, do not interpret downstream. And before comparing peak COUNTS across conditions, rarefy BAMs to a common unique-read depth (peak number scales with depth).

Pipeline Overview

FASTQ -> fastp trim -> STAR genome align -> samtools sort/index -> deepTools QC + PreSeq saturation
       -> exomePeak2 peak calling (+ MeTPeak / MACS3 cross-check)
       -> HOMER DRACH sanity check
       -> exomePeak2 differential (bam_ip + bam_treated_ip)
       -> ChIPseeker feature annotation
       -> Guitar metagene (stop-codon QC anchor) + pyGenomeTracks browser figures

Step 1: Adapter Trimming

fastp \
    --in1 raw/IP_R1.fastq.gz --in2 raw/IP_R2.fastq.gz \
    --out1 trimmed/IP_R1.fq.gz --out2 trimmed/IP_R2.fq.gz \
    --json qc/IP_fastp.json --html qc/IP_fastp.html \
    --length_required 25 --detect_adapter_for_pe --thread 8

fastp \
    --in1 raw/Input_R1.fastq.gz --in2 raw/Input_R2.fastq.gz \
    --out1 trimmed/Input_R1.fq.gz --out2 trimmed/Input_R2.fq.gz \
    --json qc/Input_fastp.json --html qc/Input_fastp.html \
    --length_required 25 --detect_adapter_for_pe --thread 8

Standard non-UMI MeRIP: do NOT pass --umi. See epitranscriptomics/merip-preprocessing for the do-NOT-dedup rationale.

Step 2: STAR Splice-Aware Genome Alignment

STAR --runMode alignReads \
    --genomeDir refs/star_index \
    --readFilesIn trimmed/IP_R1.fq.gz trimmed/IP_R2.fq.gz \
    --readFilesCommand zcat \
    --outSAMtype BAM SortedByCoordinate \
    --outFilterMultimapNmax 20 \
    --outSAMattributes NH HI AS nM NM MD \
    --outFileNamePrefix aligned/IP_rep1_ \
    --runThreadN 12

samtools index aligned/IP_rep1_Aligned.sortedByCoord.out.bam
ln -sf IP_rep1_Aligned.sortedByCoord.out.bam aligned/IP_rep1.bam     # downstream QC/peak steps consume the short ${sample}_rep${n}.bam name
ln -sf IP_rep1_Aligned.sortedByCoord.out.bam.bai aligned/IP_rep1.bam.bai

Repeat for each IP and Input replicate. Align to GENOME (not transcriptome) for downstream MeRIP peak calling. Do NOT deduplicate (no UMI in standard MeRIP).

Step 3: IP-Enrichment + Replicate-Concordance QC

multiBamSummary bins \
    --bamfiles aligned/IP_rep[0-9].bam aligned/Input_rep[0-9].bam \
    --binSize 10000 --numberOfProcessors 8 \
    -o qc/cov.npz

plotCorrelation --corData qc/cov.npz --corMethod spearman --skipZeros \
    --whatToPlot heatmap --colorMap RdYlBu_r --plotNumbers \
    -o qc/replicate_correlation.pdf

plotFingerprint \
    --bamfiles aligned/IP_rep[0-9].bam aligned/Input_rep[0-9].bam \
    --skipZeros --numberOfProcessors 8 \
    --JSDsample aligned/Input_rep1.bam \
    --outQualityMetrics qc/fingerprint_metrics.tab \
    -o qc/fingerprint.pdf

preseq lc_extrap -B -o qc/IP_rep1_lc_extrap.txt aligned/IP_rep1.bam

For peak-count comparison across conditions, rarefy BAMs to a common unique-read depth informed by the saturation curve before calling peaks.

Step 4: exomePeak2 Peak Calling (Per-Condition)

Goal: Produce a transcript-aware set of m6A peaks with FDR and IP/input fold-change from paired IP/Input genome BAM files, suitable as input to differential analysis, motif scanning, or downstream visualisation.

Approach: Build a TxDb from the matched GTF; pass paired IP/Input BAM vectors to exomePeak2() with txdb and genome (BSgenome) for GC correction; export BED12 + RDS to save_dir/.

library(exomePeak2)
library(GenomicFeatures)
library(BSgenome.Hsapiens.UCSC.hg38)

txdb <- makeTxDbFromGFF('refs/annotation.gtf', format='gtf')

result <- exomePeak2(
    bam_ip       = c('aligned/IP_rep1.bam', 'aligned/IP_rep2.bam', 'aligned/IP_rep3.bam'),
    bam_input    = c('aligned/Input_rep1.bam', 'aligned/Input_rep2.bam', 'aligned/Input_rep3.bam'),
    txdb         = txdb,
    bsgenome     = BSgenome.Hsapiens.UCSC.hg38,   # bsgenome= (a BSgenome object) for GC correction; genome= would be the UCSC string 'hg38'
    paired_end   = TRUE,
    library_type = 'unstranded',
    save_dir     = 'exomePeak2_output'            # no experiment_name arg; output goes straight under save_dir/
)

peaks <- result
nrow(peaks)   # SummarizedExomePeak has no length method (would return 1); nrow = peak count

exomePeak2() writes fixed filenames under save_dir/: Mod.bed (BED12 peaks), Mod.csv (per-peak fold-change / FDR), Mod.rds.

Step 5: MACS3 Broad-Peak Cross-Check (Optional)

macs3 callpeak \
    --treatment aligned/IP_rep[0-9].bam \
    --control aligned/Input_rep[0-9].bam \
    --format BAMPE --gsize hs \
    --nomodel --extsize 150 \
    --keep-dup all \
    --broad --broad-cutoff 0.1 --qvalue 0.05 \
    --outdir macs3_output --name m6a_run1

--keep-dup all is non-negotiable for non-UMI MeRIP (default --keep-dup 1 destroys signal at high-coverage transcripts).

Step 6: DRACH Motif Sanity Check

findMotifsGenome.pl \
    exomePeak2_output/Mod.bed \
    hg38 motif_output \
    -rna -size 100 -len 5,6 -p 8

Report DRACH enrichment on the peak set as a sanity check (P-value < 1e-50 expected). NEVER post-hoc filter individual peaks by DRACH.

Step 7: exomePeak2 Differential (Control vs Treatment)

Goal: Identify m6A peaks that change between control and treatment conditions, with per-peak log2FC + FDR, using exomePeak2's integrated peak-calling + differential interface.

Approach: Populate bam_ip + bam_input with the control arm and bam_treated_ip + bam_treated_input with the treatment arm; populating the treated arms triggers differential mode (there is NO mode= argument). Apply effect-size + FDR filters downstream.

library(exomePeak2)
library(GenomicFeatures)
library(BSgenome.Hsapiens.UCSC.hg38)

txdb <- makeTxDbFromGFF('refs/annotation.gtf', format='gtf')

ctrl_ip     <- c('aligned/ctrl_IP1.bam', 'aligned/ctrl_IP2.bam', 'aligned/ctrl_IP3.bam')
ctrl_input  <- c('aligned/ctrl_Input1.bam', 'aligned/ctrl_Input2.bam', 'aligned/ctrl_Input3.bam')
treat_ip    <- c('aligned/treat_IP1.bam', 'aligned/treat_IP2.bam', 'aligned/treat_IP3.bam')
treat_input <- c('aligned/treat_Input1.bam', 'aligned/treat_Input2.bam', 'aligned/treat_Input3.bam')

diff_result <- exomePeak2(
    bam_ip            = ctrl_ip,
    bam_input         = ctrl_input,
    bam_treated_ip    = treat_ip,
    bam_treated_input = treat_input,
    txdb              = txdb,
    bsgenome          = BSgenome.Hsapiens.UCSC.hg38,   # bsgenome=, not genome=
    paired_end        = TRUE,
    library_type      = 'unstranded',
    peak_calling_mode = 'exon',
    save_dir          = 'exomePeak2_diff_output'       # writes DiffMod.bed / DiffMod.csv; no experiment_name arg
)

diff_table <- Results(diff_result)   # SummarizedExomePeak has no as.data.frame method; Results() returns the data.frame
# differential effect-size column is DiffModLog2FC (not log2FC)
sig <- diff_table[diff_table$padj < 0.05 & abs(diff_table$DiffModLog2FC) > 0.5, ]
nrow(sig)

exomePeak2 has NO mode= or experiment_design= argument. Populating bam_treated_ip + bam_treated_input triggers differential output. For batch / antibody-lot covariate adjustment, fall through to featureCounts-on-peaks -> DESeq2 (see epitranscriptomics/m6a-differential).

Step 8: Peak Annotation to Transcript Features

library(ChIPseeker)
library(TxDb.Hsapiens.UCSC.hg38.knownGene)
library(rtracklayer)

peaks <- import('exomePeak2_output/Mod.bed')
anno <- annotatePeak(peaks, TxDb=TxDb.Hsapiens.UCSC.hg38.knownGene, level='transcript')
plotAnnoBar(anno)
plotDistToTSS(anno)

Flag peaks within ~50 nt of TSS as m6A-or-m6Am ambiguous (antibody cross-reactivity with PCIF1-deposited cap m6Am).

Step 9: Guitar Metagene (Biological QC Anchor)

library(Guitar)
library(TxDb.Hsapiens.UCSC.hg38.knownGene)

GuitarPlot(
    txTxdb          = TxDb.Hsapiens.UCSC.hg38.knownGene,
    stBedFiles      = list('exomePeak2_output/Mod.bed'),
    miscOutFilePrefix = 'figures/m6a_metagene'
)

Expected pattern: peak density rises toward and peaks near the stop codon (3'UTR-proximal end of CDS). If absent, suspect IP failure or wrong antibody; do NOT proceed to downstream interpretation.

Complete Bash Driver

#!/usr/bin/env bash
set -euo pipefail

STAR_INDEX=$1
GTF=$2
IP_R1=$3
IP_R2=$4
INPUT_R1=$5
INPUT_R2=$6
OUTPUT_DIR=$7

mkdir -p "${OUTPUT_DIR}"/{qc,trimmed,aligned,peaks,figures}

fastp --in1 "${IP_R1}" --in2 "${IP_R2}" \
    --out1 "${OUTPUT_DIR}/trimmed/IP_R1.fq.gz" --out2 "${OUTPUT_DIR}/trimmed/IP_R2.fq.gz" \
    --json "${OUTPUT_DIR}/qc/IP_fastp.json" --length_required 25 --detect_adapter_for_pe --thread 8

fastp --in1 "${INPUT_R1}" --in2 "${INPUT_R2}" \
    --out1 "${OUTPUT_DIR}/trimmed/Input_R1.fq.gz" --out2 "${OUTPUT_DIR}/trimmed/Input_R2.fq.gz" \
    --json "${OUTPUT_DIR}/qc/Input_fastp.json" --length_required 25 --detect_adapter_for_pe --thread 8

for sample in IP Input; do
    STAR --runMode alignReads --genomeDir "${STAR_INDEX}" \
        --readFilesIn "${OUTPUT_DIR}/trimmed/${sample}_R1.fq.gz" "${OUTPUT_DIR}/trimmed/${sample}_R2.fq.gz" \
        --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate \
        --outFilterMultimapNmax 20 \
        --outFileNamePrefix "${OUTPUT_DIR}/aligned/${sample}_" --runThreadN 12
    samtools index "${OUTPUT_DIR}/aligned/${sample}_Aligned.sortedByCoord.out.bam"
done

macs3 callpeak \
    --treatment "${OUTPUT_DIR}/aligned/IP_Aligned.sortedByCoord.out.bam" \
    --control "${OUTPUT_DIR}/aligned/Input_Aligned.sortedByCoord.out.bam" \
    --format BAMPE --gsize hs --nomodel --extsize 150 --keep-dup all \
    --broad --broad-cutoff 0.1 --qvalue 0.05 \
    --outdir "${OUTPUT_DIR}/peaks" --name m6a

The full pipeline (incl. exomePeak2 peak calling, DRACH check, ChIPseeker annotation, Guitar metagene) is best orchestrated in Snakemake or Nextflow with the per-skill recipes from the four epitranscriptomics/ skills.

QC Checkpoints

| Checkpoint | Expected | Action if Failed | |------------|----------|------------------| | Properly-paired rate (samtools flagstat) | >=85% | Check trimming and adapter contamination | | Replicate Spearman within condition (10 kb bins) | >=0.85 IP-IP | Inspect divergent replicate; consider exclusion | | plotFingerprint IP-vs-input JS distance | >=0.5 | Suspect failed IP if lower | | Saturation plateau depth | ~30-60M unique reads | Sequence deeper if not plateaued | | DRACH motif enrichment (HOMER, peak set) | P-value < 1e-50 | Suspect IP failure or wrong antibody | | Stop-codon enrichment in Guitar metagene | Clear 3'UTR-proximal peak | Suspect IP failure, wrong antibody, or non-m6A modification | | 5'UTR peaks fraction | Note ambiguity zone (~50 nt of TSS) | Flag as m6A-or-m6Am ambiguous; PCIF1 cross-reactivity |

Output Files

| File | Description | |------|-------------| | exomePeak2_output/Mod.bed | exomePeak2 peak BED12 | | exomePeak2_diff_output/DiffMod.bed | Differential peaks with log2FC + FDR | | motif_output/ | HOMER DRACH motif enrichment report | | figures/m6a_metagene.pdf | Guitar transcript-feature metagene (stop-codon QC anchor) | | qc/replicate_correlation.pdf | deepTools Spearman heatmap | | qc/fingerprint.pdf | deepTools Lorenz IP-enrichment plot | | qc/IP_rep*_lc_extrap.txt | PreSeq saturation curves |

Common Errors

| Symptom | Cause | Fix | |---------|-------|-----| | Strongest m6A peaks (high-expression transcripts) vanish | Deduplicated non-UMI MeRIP, or MACS3 default --keep-dup 1 | Keep all reads (--keep-dup all); never dedup non-UMI MeRIP | | exomePeak2 runs but gives no differential output | Expected a mode=/experiment_design= argument | Populate bam_treated_ip + bam_treated_input to trigger differential | | Real non-canonical m6A sites lost | Filtered individual peaks by DRACH presence | DRACH is a peak-SET sanity check (E<1e-50), never a per-peak filter | | Peak counts "differ" between conditions but it's depth | Compared raw peak numbers at unequal depth | Rarefy BAMs to a common unique-read depth before cross-condition counts | | No stop-codon enrichment in the metagene | IP failure, wrong antibody, or non-m6A signal | STOP; do not interpret downstream (Guitar go/no-go) | | 5'UTR peaks over-interpreted as m6A | Antibody cross-reacts with cap-adjacent m6Am (PCIF1) | Flag peaks within ~50 nt of TSS as m6A-or-m6Am ambiguous |

References

• Dominissini D, Moshitch-Moshkovitz S, Schwartz S, et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201-206. DOI 10.1038/nature11112. (MeRIP/m6A-seq; stop-codon enrichment.)
• Meyer KD, Saletore Y, Zumbo P, et al (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell 149:1635-1646. DOI 10.1016/j.cell.2012.05.003.
• Meng J, Lu Z, Liu H, et al (2014) A protocol for RNA methylation differential analysis with MeRIP-Seq data and the exomePeak R/Bioconductor package. Methods 69:274-281. DOI 10.1016/j.ymeth.2014.06.008. (exome-based peak calling.)
• Cui X, Wei Z, Zhang L, et al (2016) Guitar: an R/Bioconductor package for gene annotation guided transcriptomic analysis of RNA-related genomic features. BioMed Research International 2016:8367534. DOI 10.1155/2016/8367534. (transcript-feature metagene.)

Related Skills

• epitranscriptomics/merip-preprocessing - Per-step preprocessing (trim, align, QC, saturation, IP-over-Input bigWig)
• epitranscriptomics/m6a-peak-calling - exomePeak2 / MeTPeak / MACS3 deep treatment, DRACH sanity check, m6A-vs-m6Am 5'UTR flag
• epitranscriptomics/m6a-differential - Differential methods (exomePeak2, QNB, RADAR), batch / lot covariate handling, stoichiometry-vs-expression confound
• epitranscriptomics/modification-visualization - Guitar metagene, peak-centred heatmaps, pyGenomeTracks browser figures
• epitranscriptomics/m6anet-analysis - ONT direct-RNA alternative for orthogonal stoichiometry validation
• chip-seq/peak-calling - Sibling IP-vs-input peak-calling framework
• chip-seq/chipseq-qc - IP enrichment QC concepts that transfer to MeRIP
• read-alignment/star-alignment - General STAR splice-aware alignment
• workflow-management/snakemake-workflows - Snakemake orchestration patterns
• workflow-management/nextflow-pipelines - Nextflow orchestration patterns
• workflows/rnaseq-to-de - General RNA-seq -> DE pipeline patterns