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BIOF501_term_project
Differential m6A RNA methylation with Direct RNA sequencing
1. Background
N6-methyladenosine (m6A) is the most common RNA modifications modulating functions of cellular RNA species.
xPore is a Python package identifying differentail RNA modifications from Nanopore sequencing data.
xPore can identify positions of m6A sites at single-base resolution.
2. Dataset Description
Demo samples are downloaded from link provided by xPore.
METTL3 is the most dominant m6A methylation writter.
HEK293T-METTL3-KO-rep1
is METTL3 knockout (KO) of HEK293T cell line.
(m6a_test/HEK293T-METTL3-KO-rep1/fastq/)
HEK293T-WT-rep1
is wild-type (WT) of HEK293T cell line.
(m6a_test/HEK293T-WT-rep1/fastq/)
By comparing two different conditions, xPore will run differnetial m6a methylation analysis.
3. Workflow Visualization
-
Align to transcriptome
-
Bam files indexing
-
Resquiggle using
nanopolish -
xPoredifferentail RNA modifications analysis
4. Results
Result table will be generated
m6a_test/out/diffmod.table
Result table shown below:
id position kmer diff_mod_rate_KO_vs_WT pval_KO_vs_WT z_score_KO_vs_WT ... sigma2_unmod sigma2_mod conf_mu_unmod conf_mu_mod mod_assignment t-test
ENSG00000114125 141745412 GGACT -0.823318 4.241373e-115 -22.803411 ... 5.925238 18.048687 0.968689 0.195429 lower 1.768910e-19
ENSG00000159111 47824212 GGACT -0.828023 1.103790e-88 -19.965293 ... 2.686549 13.820089 0.644436 0.464059 lower 5.803242e-18
ENSG00000159111 47824138 GGGAC -0.757891 1.898161e-73 -18.128515 ... 3.965195 9.877299 0.861480 0.359984 lower 9.708552e-08
5. Cloning
Clone this repository and also submodules not manageable by conda(or pip)
git clone --recursive https://github.com/sunsetyerin/BIOF501_term_project.git
6. Python 3 environment
This repository has been developped using python3.7.3 through python3.7.6 in a miniconda environment.
Though conda is not required, it is highly recommended in order to manage the dependencies.
Using snakemake to manage the environment
From a bash terminal:
snakemake --use-conda --conda-create-envs-only --cores [cores available]
7. Snakemake usage of this repo
All scripts, steps and jobs are managed using snakemake
Input files, workflow parameters and output path are provided through config
files (
configs/sample_config.yaml
).
The workflow is designed to run differentially methylated m6A RNA analysis with different conditions
# To display the jobs and commands including subworkflows
snakemake general -np
# to visualize the acyclic rulegraph
snakemake general --forceall --rulegraph | dot -Tpdf > dag.pdf
# To launch the jobs
snakemake general --use-conda
8. Perform the processing of a single sample
Config files contains the values of variables needed to perform Differential RNA modifications analysis.
Most of the data directories hosting results will be created when necessary by the snakemake manager, although it is possible to create the folders in advance.
Code Snippets
49 50 51 52 53 54 55 | shell: " ".join(["minimap2 -ax map-ont -uf -t {resources.cpus} --secondary=no", "{params.ref} {input.fastq} > {output.aligned}", "2>> {log}"]) rule sam2bam: """ |
78 79 80 81 82 | shell: " ".join(["samtools view -Sb {input.sam} | samtools sort -o {output.bam} - &>> {log}"]) rule bamidx: """ |
105 106 107 108 109 | shell: " ".join(["samtools index {input.bam_sort} > {output.bamidx} &>> {log}"]) rule nanopolish_index: """ |
134 135 136 137 138 139 140 141 | shell: " ".join(["nanopolish index", "--directory {input.fast5_dir} {input.fastq} --verbose", # "> {output.nanopolish_index}", "&> >(tee {log})"]) rule nanopolish_eventalign: """ |
181 182 183 184 185 186 187 188 189 190 191 192 193 | shell: " ".join(["nanopolish eventalign", "--reads {input.fastq}", "--bam {input.bam_sort}", "--genome {input.genome}", "--signal-index", "--scale-events", "--threads {resources.cpus}", "> {output.nanopolish_eventalign}", "&> >(tee {log})"]) rule xpore_dataprep: """ |
222 223 224 225 226 227 228 229 230 231 232 233 | shell: " ".join(["xpore dataprep", "--eventalign {input.nanopolish_eventalign}", #"--gtf_or_gff {input.genomic_annotation}", #"--transcript_fasta {input.transcriptome}", "--out_dir {params.outdir}", #"--genome", "--n_processes {resources.cpus}", "&> >(tee {log})"]) rule xpore_diffmode: """ |
257 258 | shell: "xpore diffmod --config {input.xpore_config} --n_processes {resources.cpus} 2> {log}" |
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