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Modkit pile up Inconsistent biological results for RNA m5c

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#628 10 comentarios 0 reacciones 0 asignados Ver en GitHub

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Evaluación

Dificultad
4/5
Tiempo estimado
3-5 días
Aptitud para principiantes
35/100
Tipo de issue
Error
Claridad
Necesita aclaración
Estado de actividad
Tranquilo
Stack tecnológico
rust, shell
Área
bioinformatics, cli

Línea de trabajo

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.

Escrito por el modelo de indexación a partir del texto del issue.

Descripción

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

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Lenguaje dominante
Rust
Estrellas
274
Forks
33
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