ModPhred is a pipeline for detection, annotation and visualisation of DNA/RNA modifications (From Authors' documentation)..

References:

• Pryszcz LP and Novoa EM ModPhred: an integrative toolkit for the analysis and storage of nanopore sequencing DNA and RNA modification data. Bioinformatics, 38:257-260 (2022)

• Modphred Main Site: ReadTheDocs

• Module Name: modphred (see the modules page for more information)
• You will need to request GPU resources to run modphred (see example below). the current version of modphred will not work on a100 GPUs

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --gres=gpu:p100:1,lscratch:200 --mem=16g --cpus-per-task=6
salloc.exe: Pending job allocation 46116226
salloc.exe: job 46116226 queued and waiting for resources
salloc.exe: job 46116226 has been allocated resources
salloc.exe: Granted job allocation 46116226
salloc.exe: Waiting for resource configuration
salloc.exe: Nodes cn4224 are ready for job

[user@cn4224 ~]$ export TMPDIR=/lscratch/${SLURM_JOB_ID}
[user@cn4224 ~]$ module load modphred
[user@cn4224 ~]$ cd /data/${USER}
[user@cn4224 ~]$ wget https://public-docs.crg.es/enovoa/public/lpryszcz/src/modPhred/test/ -q -r -c -nc -np -nH --cut-dirs=6 --reject="index.html*"
[user@cn4224 ~]$ run -f ref/ECOLI.fa -o OUTPUT -i PRJEB22772/* -t4 --host /usr/bin/guppy_basecall_server
[2022-12-29 11:34:36] ===== Welcome, welcome to modPhred pipeline! =====
[2022-12-29 11:34:36] Starting /usr/bin/guppy_basecall_server ... [mem:   134 MB]
[2022-12-29 11:34:40] Encoding modification info from 2 directories... [mem:   134 MB]
[2022-12-29 11:34:40]  PRJEB22772/MARC_ZFscreens_R9.4_1D-Ecoli-run_FAF05145 with 4 Fast5 file(s)... [mem:   134 MB]
[2022-12-29 11:36:21]   DNA alphabet with 2 modification(s) {'A': ['Y'], 'C': ['Z'], 'G': [], 'T': []}. symbol2modbase: {'Y': '6mA', 'Z': '5mC'}
[2022-12-29 11:39:20]   106,722,317 bases saved in FastQ, of those: 332,925 6mA [ 0.312%], 160,288 5mC [ 0.150%]   
[2022-12-29 11:39:20]  PRJEB22772/MARC_ZFscreens_R9.4_2D-Ecoli-run_FAF05711 with 1 Fast5 file(s)... [mem:   988 MB]
[2022-12-29 11:41:40]   DNA alphabet with 2 modification(s) {'A': ['Y'], 'C': ['Z'], 'G': [], 'T': []}. symbol2modbase: {'Y': '6mA', 'Z': '5mC'}
[2022-12-29 11:41:40]   29,420,071 bases saved in FastQ, of those: 91,315 6mA [ 0.310%], 51,017 5mC [ 0.173%]   
[2022-12-29 11:41:40] Aligning FastQ files from 2 directories... [mem:  2292 MB]
[2022-12-29 11:41:40]   > OUTPUT/minimap2/MARC_ZFscreens_R9.4_1D-Ecoli-run_FAF05145.bam [mem:  2292 MB]
[2022-12-29 11:42:01]   > OUTPUT/minimap2/MARC_ZFscreens_R9.4_2D-Ecoli-run_FAF05711.bam [mem:  2292 MB]
[2022-12-29 11:42:09] Indexing bam file(s)... [mem:  2392 MB]
[2022-12-29 11:42:11] Reporting positions that are likely modified to OUTPUT/mod.gz ... [mem:  2392 MB]
[2022-12-29 11:42:11]  Getting regions covered by at least 25 reads... [mem:  2392 MB]
[2022-12-29 11:42:12]   7 regions to process... [mem:  2392 MB]
[2022-12-29 11:45:50] Loading modification data... [mem:  2392 MB]
[2022-12-29 11:45:50] Plotting... [mem:  2392 MB]
[2022-12-29 11:45:51] Saving modified positions with max frequency as OUTPUT/mod.bed (bedMethyl file) ... [mem:  2392 MB]
[2022-12-29 11:45:51]  and separately for every BAM file as OUTPUT/minimap2/*.bed ... [mem:  2392 MB]
[2022-12-29 11:46:00] Saving plots for depth, basecall_accuracy, mod_frequency, median_mod_prob to OUTPUT/plots ... [mem:  2392 MB]
[2022-12-29 11:46:05] You can remove reads directory: rm -r OUTPUT/reads/ [mem:  2392 MB]
[2022-12-29 11:46:05] All finished! Have a nice day :) [mem:  2392 MB]
#Time elapsed: 0:11:28.435555

[user@cn4224 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

Create a batch input file (e.g. modphred.sh) similar to the following.

#! /bin/bash

module load modphred
run -f ref/ECOLI.fa -o OUTPUT -i PRJEB22772/* -t4 --host /usr/bin/guppy_basecall_server

Submit these jobs using the Slurm sbatch command:

sbatch --partition=gpu --cpus-per-task=6 --mem=16g --gres=lscratch:200,gpu:p100:1 modphred.sh

Create a swarmfile for the pipeline (e.g. modphred.swarm). For example:

run -f ref/ECOLI.fa -o OUTPUT2 -i PRJEB22772/* -t4 --host /usr/bin/guppy_basecall_server
run -f ref/ECOLI.fa -o OUTPUT3 -i PRJEB22773/* -t4 --host /usr/bin/guppy_basecall_server
run -f ref/ECOLI.fa -o OUTPUT4 -i PRJEB22774/* -t4 --host /usr/bin/guppy_basecall_server

Submit this job using the swarm command.

swarm -f modphred.swarm --partition=gpu -g 16 -t 6 --gres=gpu:p100:1,lscratch:200 --module modphred