Hacktoberfest 2026: le issue che i maintainer hanno segnato per ottobre, aperte e adatte ai principianti. Sfoglia le issue Hacktoberfest

Modkit pile up Inconsistent biological results for RNA m5c

Aperta
  • Qualcosa non va?
#628 10 commenti 0 reazioni 0 assegnatari Vedi su GitHub

Nessuno ha ancora preso questa issue.

Valutazione

Difficoltà
4/5
Tempo stimato
3-5 giorni
Idoneità per principianti
35/100
Tipo di issue
Bug
Chiarezza
Da chiarire
Stato di attività
Tranquilla
Stack tecnologico
rust, shell
Ambito
bioinformatics, cli

Direzione di ricerca

Start with the modkit pileup and dmr pair commands in the report, then inspect the referenced pileup and DMR log files alongside the threshold and coverage options. Compare the control and NSUN2 KD inputs and the resulting stoichiometry and differential calls; the issue is done when the cause of the unexpected KD results is identified or a reproducible modkit problem is isolated.

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

Descrizione

Analysis Question

Hi everyone,
first time posting an issue. First thank you for having developed and keeping updating modkit, extremely useful for my research. Indeed, I am interested in RNA m5c methylation. I have ONT generated data from 2 cell lines, namely a control condition and a line knockdown (KD) for NSUN2, a major m5c methylator. In particular I added the latter as negative and quality control, to be sure that the called m5c sites disappeared upon NSUN2 KD. I have used the following lined of codes for modkit pileup

$modkit pileup
-t "${SLURM_CPUS_PER_TASK}"
--filter-threshold 0.8
--mod-threshold C:0.99
--modified-bases C:m
--reference "$REF" \ # alignment to genome
"${input_bam}"
"${output_dir}/${sample_name}.raw.bed"
--log-filepath "${output_dir}/${sample_name}.pileup.log"

Then I use an inhouse R script for differential methylation analysis as well as modkit dmr pair with the following commands

$modkit dmr pair
-a "$STG/Pilot_sample1/Pilot_sample1.raw.bed.gz" . # a CTRL condition
-a "$STG/Pilot_sample2/Pilot_sample2.raw.bed.gz"
-b "$STG/Pilot_sample5/Pilot_sample5.raw.bed.gz" \ # B NSUN2 KD
-b "$STG/Pilot_sample6/Pilot_sample6.raw.bed.gz"
--ref "$REF"
--base C
--single-code m
--cap-coverages
--min-valid-coverage 20
--header
-o "$DMR/control_vs_siNSUN2.dmr.bed"
--log-filepath "$DMR/control_vs_siNSUN2.dmr.log"
-t "${SLURM_CPUS_PER_TASK}"

I have been trying with multiple combination of thresholds but I keep getting thos kinds of results (the plot shows the significantly differentially methylated m5c sites between NSUN2 KD vs CTRL plotted according to the stoichiometry in the CTRL condition). From the biological perspective, It unlikely that I have more site in the NSUN2 KD condition, so I don t know what could have gone wrong (biologically I am sure of the knockdown of the protein, checked by different other analysis).
Could you help me? Thanks

Image

Marcello

Lingua principale
Rust
Stelle
274
Fork
33
Nessuna PR unita negli ultimi 30g

Preparare l'ambiente

Non abbiamo ancora controllato i file di configurazione di questo progetto. Parti dal suo README e consulta la nostra guida al primo contributo per i passaggi generali.

Come iniziare

  1. Leggi tutta la issue e poi la guida ai contributi del progetto.
  2. Commenta sulla issue per dire che te ne occupi tu — evita che due persone facciano lo stesso lavoro.
  3. Fai un fork del repository e lavora su un branch.
  4. Apri una pull request che faccia riferimento al numero della issue.

Altre issue di nanoporetech/modkit

Tutte le issue di nanoporetech/modkit

Issue simili

Altre issue su Rust

Ricevi le nuove issue nella tua casella

Un breve riepilogo di issue GitHub adatte ai principianti.