Usage	Requirement

TABLE OF CONTENTS

• AIM
• WARNINGS
• CONTENT
• HOW TO RUN
• OUTPUT
• VERSIONS
• LICENCE
• CITATION
• CREDITS
• ACKNOWLEDGEMENTS
• WHAT'S NEW IN

AIM

Return homopolymers info per DNA sequence in a batch of DNA sequences, as well as statistics about homopolymers for this batch.

WARNINGS

The algorithm works as if it splits the input sequence according to homopolymers and then returns the info.

With the input sequence ATTTAAGCGGG, the homopolymers are:
A
TTT
AA
G
C
GGG

CONTENT

Files and folder	Description
main.nf	File that can be executed using a linux terminal, a MacOS terminal or Windows 10 WSL2.
nextflow.config	Parameter settings for the main.nf file. Users have to open this file, set the desired settings and save these modifications before execution.
bin folder	Contains files required by the main.nf file.
Licence.txt	Licence of the release.

INPUT

Required files
A fasta file.

The dataset used in the nextflow.config file, as example, is available at https://zenodo.org/records/10681460.

File name	Description
test.fasta	Fasta file . Available here.

HOW TO RUN

1. Prerequisite

Installation of:
nextflow DSL2
Graphviz, sudo apt install graphviz for Linux ubuntu
Apptainer

2. Local running (personal computer)

2.1. main.nf file in the personal computer

• Mount a server if required:

DRIVE="Z" # change the letter to fit the correct drive
sudo mkdir /mnt/share
sudo mount -t drvfs $DRIVE: /mnt/share

Warning: if no mounting, it is possible that nextflow does nothing, or displays a message like:

Launching `main.nf` [loving_morse] - revision: d5aabe528b
/mnt/share/Users

• Run the following command from where the main.nf and nextflow.config files are (example: \wsl$\Ubuntu-20.04\home\gael):

nextflow run main.nf -c nextflow.config

with -c to specify the name of the config file used.

2.2. main.nf file in the public git repository

Run the following command from where you want the results:

nextflow run gael-millot/homopolymer # github, or nextflow run http://github.com/gael-millot/homopolymer
nextflow run -hub pasteur gmillot/homopolymer -r v1.0.0 # gitlab

3. Distant running (example with the Pasteur cluster)

3.1. Pre-execution

Copy-paste this after having modified the EXEC_PATH variable:

EXEC_PATH="/pasteur/zeus/projets/p01/BioIT/gmillot/homopolymer" # where the bin folder of the main.nf script is located
export CONF_BEFORE=/opt/gensoft/exe # on maestro

export JAVA_CONF=java/13.0.2
export JAVA_CONF_AFTER=bin/java # on maestro
export APP_CONF=apptainer/1.3.5
export APP_CONF_AFTER=bin/apptainer # on maestro
export GIT_CONF=git/2.39.1
export GIT_CONF_AFTER=bin/git # on maestro
export GRAPHVIZ_CONF=graphviz/2.42.3
export GRAPHVIZ_CONF_AFTER=bin/graphviz # on maestro

MODULES="${CONF_BEFORE}/${JAVA_CONF}/${JAVA_CONF_AFTER},${CONF_BEFORE}/${APP_CONF}/${APP_CONF_AFTER},${CONF_BEFORE}/${GIT_CONF}/${GIT_CONF_AFTER}/${GRAPHVIZ_CONF}/${GRAPHVIZ_CONF_AFTER}"
cd ${EXEC_PATH}
chmod 755 ${EXEC_PATH}/bin/*.*
module load ${JAVA_CONF} ${APP_CONF} ${GIT_CONF} ${GRAPHVIZ_CONF}

3.2. main.nf file in a cluster folder

Modify the second line of the code below, and run from where the main.nf and nextflow.config files are (which has been set thanks to the EXEC_PATH variable above):

HOME_INI=$HOME
HOME="${HELIXHOME}/homopolymer/" # $HOME changed to allow the creation of .nextflow into /$HELIXHOME/homopolymer/, for instance. See NFX_HOME in the nextflow software script
nextflow run main.nf -c nextflow.config
HOME=$HOME_INI

3.3. main.nf file in the public git repository

Modify the first and third lines of the code below, and run (results will be where the EXEC_PATH variable has been set above):

VERSION="v1.0"
HOME_INI=$HOME
HOME="${HELIXHOME}/homopolymer/" # $HOME changed to allow the creation of .nextflow into /$HELIXHOME/homopolymer/, for instance. See NFX_HOME in the nextflow software script
nextflow run gael-millot/homopolymer -r $VERSION -c $HOME/nextflow.config #github, or nextflow run http://github.com/gael-millot/homopolymer -r $VERSION -c $HOME/nextflow.config
nextflow run -hub pasteur gmillot/homopolymer -r $VERSION -c $HOME/nextflow.config # gitlab
HOME=$HOME_INI

4. Error messages and solutions

Message 1

Unknown error accessing project `gmillot/homopolymer` -- Repository may be corrupted: /pasteur/sonic/homes/gmillot/.nextflow/assets/gmillot/homopolymer

Purge using:

rm -rf /pasteur/sonic/homes/gmillot/.nextflow/assets/gmillot*

Message 2

WARN: Cannot read project manifest -- Cause: Remote resource not found: https://gitlab.pasteur.fr/api/v4/projects/gmillot%2Fhomopolymer

Contact Gael Millot (distant repository is not public).

Message 3

permission denied

Use chmod to change the user rights. Example linked to files in the bin folder:

chmod 755 bin/*.*

OUTPUT

An example of results is present at this address: https://zenodo.org/records/10681595/files/homopolymer_2_1708386120.zip.

homopolymer_ <HOMOPOLYMER_MIN_LENGTH>_ <UNIQUE_ID> folder	Description
report.html	Report of the analysis.
reports	Folder containing all the reports of the different processes, including the nextflow.config file used.
figures	Folder containing the graphs in png format that are used in the report.html file, as well as the corresponding svg vectorial files if needed.
files	Folder containing the following files: homopol_summary.tsv Column description: Warning: columns takes into account the min_length parameter in the nextflow.config file, meaning that the results are only for homopolymer lengths equal or above min_length. But, the two homopol_obs_distrib and homopol_theo_distrib columns provides the distribution of all the polymers, whatever min_length. name: name of the input sequence of the batch. seq_length: nb of bases in the input sequence. max_homopol_size: number of times the nucleotide is repeated in the longest homopolymer (i.e., length). If several longest homopolymers in the input sequence, results are semi-colon separated in each cell. nucleotide: nucleotide of the homopolymer of max_homopol_size. If several longest homopolymers in the input sequence, results are semi-colon separated in each cell. starting_position: position of the first nucleotide of the homopolymer of max_homopol_size. If several longest homopolymers in the input sequence, results are semi-colon separated in each cell. relative_position: relative position of the starting_position value in the input sequence when the first base of the sequence is 0 and the last one is 1. The formula used is y = 0 if seq_length is 1 and y = (starting_position - 1) / seq_length otherwise, to get y between 0 and (starting_position - 1) / seq_length (i.e., not 1). If several longest homopolymers in the input sequence, results are semi-colon separated in each cell. nb: number of consecutive homopolymers in the input sequence. mean_size: average homopolymer size among the number of consecutive homopolymers in the input sequence (not considering the homopolymers below the min_length parameter in the nextflow.config file, meaning that the mean is computed only on the length of the considered homopolymers, not using the whole input sequence length). homopol_obs_distrib: number of homopol of size 1, 2, ..., n (semi-colon separator). Warning: the min_length parameter in the nextflow.config is ignored. homopol_theo_distrib: number of homopol of size 1, 2, ..., n (semi-colon separator). Warning: the min_length parameter in the nextflow.config is ignored. boxplot_stat.tsv Column description: From homopol_obs_distrib and homopol_theo_distrib columns of the homopol_summary.tsv file. length: homopolymer length. freq: frequency (sum of all the homopolymer numbers of size length in all the input sequences. kind: observed or random (theoretical) homopolymers. scatterplot_stat.tsv Column description: From homopol_obs_distrib and homopol_theo_distrib columns of the homopol_summary.tsv file. length: homopolymer length. kind: observed or random (theoretical) homopolymers. mean: frequency mean along all the sequences of the batch (sum of the number of homopolymers of size categ in each input sequence) / (number of sequences). sd: frequency standard deviation along all the sequences of the batch (sd of the corresponding mean). CI95.inf: 95% lower Confidence Interval of the mean, according to the normal law(mean, sd). CI95.sup: 5% upper Confidence Interval of the mean, according to the normal law(mean, sd). plot_raw_values.tsv Column description: From homopol_obs_distrib and homopol_theo_distrib columns of the homopol_summary.tsv file. Each line is a proportion of one polymer length in one sequence. The total nomber of rows should be: number of different homopolymer lengths x number of sequences x number of kind. prop: proportion of one polymer length in one sequence. length: homopolymer length. gene_name: name of the sequence. kind: observed or random (theoretical) homopolymers. t_test.tsv Column description: The t test table displayed in the report.html file. length: homopolymer length. obs.mean: mean of the observed homopolymers in the batch of sequences. theo.mean: mean of the random homopolymers. obs.sd: standard deviation of the observed homopolymers in the batch of sequences. theo.sd: standard deviation of the random homopolymers. df: degree of freedom of the t test. t: t test statistics. p.value: p value. BH.adj.p.value: Benjamini Hochberg adjusted p values along all the t tests performed. graph_stat.RData .RData file containing all the objects used to make the report.html, that can be reused if necessary.

VERSIONS

The different releases are tagged here.

LICENCE

This package of scripts can be redistributed and/or modified under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. Distributed in the hope that it will be useful, but without any warranty; without even the implied warranty of merchandability or fitness for a particular purpose. See the GNU General Public License for more details at https://www.gnu.org/licenses or in the Licence.txt attached file.

CITATION

Not yet published.

CREDITS

Gael A. Millot, Hub, Institut Pasteur, Paris, France.

ACKNOWLEDGEMENTS

The developers & maintainers of the mentioned softwares and packages, including:

• R
• ggplot2
• Nextflow
• Apptainer
• Docker
• Git
• Github
• Gitlab
• Bash
• Ubuntu

Special acknowledgement to Yoann Dufresne, Hub, Institut Pasteur, Paris, France.

WHAT'S NEW IN

5.4

• In the nextflow.config file, upgrade singularity -> apptainer (real one).

5.3

• In the nextflow.config file, upgrade singularity -> apptainer.

v5.2

• Bugs fixed in report.
• README improved for github.
• nextflow.config input from zenodo.

v5.1

Bug fixed in report.

v5.0

Nextflow DSL1 -> DSL2.

v4.3

Plot_raw_values.tsv file added and boxplot_stat_log.tsv file modified.

v4.2

Plot and html report modified.

v4.1

Boxplots modified.

v4.0

Completemy rewritten.

v3.2

Minimum length of homopolymer added as parameter, among other things.

v3.1

Many things improved.

v3.0

Completely modified. Now the file is a nextflow and outputs include tables ,graphs and stats.

v2.0

New features included in the result table.

v1.0

Everything.