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`pileup` command is giving BED files that `dmr` command says `invalid-bedmethyl-data`.

Aperta
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Valutazione

Difficoltà
4/5
Tempo stimato
3-5 giorni
Idoneità per principianti
42/100
Tipo di issue
Bug
Chiarezza
Abbastanza chiara
Stato di attività
Ferma
Stack tecnologico
rust
Ambito
bioinformatics, cli

Direzione di ricerca

Start by comparing the BED-methyl output from modkit pileup with the record rejected by modkit dmr pair. Inspect modkit-core/src/pileup/subcommand.rs and the DMR validation path indicated by the invalid-bedmethyl-data error; use the supplied commands and logs as the reproduction. Done means pileup output is accepted by dmr without the reported coverage/count inconsistency.

Scritto dal modello di indicizzazione a partire dal testo della issue.

Descrizione

troubleshooting

pileup command is giving BED files that dmr command says invalid-bedmethyl-data. I have attached the output of DMR and the commands I have run.

OUTPUT (DMR)

> reading reference FASTA at "<genome_fasta_name>.fasta"
> 1521 common sequence(s) between FASTA and both samples
> running single-site analysis
> using default prior, Beta(α: 0.55, β: 0.55)
> estimating max coverages from data
> sampled 1094523 a records and 905404 b records, calculating max coverages for 95th percentile
> calculated max coverage for a: 6 and b: 21
> running with replicates, but not matched samples
> batch failed: invalid data, valid coverage (1) is not equal to the sum of canonical and modified counts (0), [BedMethylLine { chrom: "AAKM01000036", interval: Interval { start: 7056, stop: 7057, val: () }, raw_mod_code: Code('h'), strand: Positive, count_methylated: 0, valid_coverage: 1, count_canonical: 0, count_other: 1, count_delete: 0, count_fail: 0, count_diff: 0, count_nocall: 0 }] chrom: AAKM01000036 starting at 7056, stopping
> 12 batches processed                                                                                                                                                                 > Error! invalid-bedmethyl-data

PILEUP LOGS

[modkit-logging/src/lib.rs::69][2026-02-07 10:41:20][DEBUG] command line: modkit pileup J_CC_5hmC.bam J_CC_5hmC.bed.gz --modified-bases 5hmC --reference /home/<genome_fasta_name>.fasta --log J_CC_5hmC.log --bgzf
[modkit-core/src/pileup/subcommand.rs::802][2026-02-07 10:41:20][INFO] discarded 1107 contigs with zero aligned reads
[modkit-core/src/pileup/subcommand.rs::576][2026-02-07 10:41:20][INFO] parsed 1 base modification(s). Base modifications other than 'C:h' will be counted as 'N_other'.
[modkit-core/src/pileup/subcommand.rs::653][2026-02-07 10:41:20][INFO] adding single-base motif: 'C 0'
[modkit-core/src/pileup/subcommand.rs::944][2026-02-07 10:41:20][INFO] using bgzf compression with 4 compression threads
[modkit-core/src/command_utils.rs::102][2026-02-07 10:41:20][INFO] attempting to sample 10042 reads
[modkit-core/src/reads_sampler/mod.rs::49][2026-02-07 10:41:20][DEBUG] found BAM index, sampling reads in 1000000 base pair chunks
[modkit-core/src/reads_sampler/sampling_schedule.rs::261][2026-02-07 10:41:20][DEBUG] removed 0 contigs from schedule with <= 1 reads
[modkit-core/src/reads_sampler/sampling_schedule.rs::141][2026-02-07 10:41:20][DEBUG] derived sampling schedule, sampling total 11547 reads from 1641 contigs, 0 unmapped reads
[modkit-core/src/reads_sampler/sampling_schedule.rs::164][2026-02-07 10:41:20][DEBUG] schedule
 chrom  count/frac

DMR Command

modkit dmr pair \
  -a UNC_ED_5hmC.bed.gz \
  -a UNC_YR1_5hmC.bed.gz \
  -a UNC_YR2_5hmC.bed.gz \
  -a UNC_EL_5hmC.bed.gz \
  -a UNC_FY_5hmC.bed.gz \
  -b CM_EL_5hmC.bed.gz \
  -b CM_EJ_5hmC.bed.gz \
  -o DMR_UNC_vs_CM_5hmC.bed \
  --ref "${REF}" \
  --base C \
  --threads "${THREADS}" \
  --log-filepath DMR_UNC_vs_CM_5hmC.log

PILEUP Command. mod = 5hmC

pileup () {
    local bam=$1
    local mod=$2
    local prefix=$3
    log "Running modkit pileup (${mod}) for ${prefix}"
    modkit pileup \
        "${bam}" \
        "${prefix}_${mod}.bed.gz" \
        --modified-bases "${mod}" \
        --reference "${REF}" \
        --log "${prefix}_${mod}.log" \
        --bgzf
}

I had run modified basecalling for 5mC 5hmC to create modified BAM files. @ArtRand

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