TOP

Contacts

• Christine Pourcel ([email protected])
• Jean-Philippe Vernadet ([email protected])

The TOP (Transcript Orientation Pipeline) pipeline was initially designed to determine the orientation of CRISPR sequences from transcripts from high throughput sequencing. This pipeline would improve the database [CRISPRCasdb]. However, it could be used to determine the orientation of any sequence of interest.

Installation

• Conda

# Get miniconda 
wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh;
./Miniconda3-latest-Linux-x86_64.sh

• TOP environment and scripts

# Clone or download files
git clone https://github.com/i2bc/TOP.git

# Create TOP_env
conda env create -f Scripts/TOP.yaml -n TOP_env

How to use

• Preparation of the required sequences/archives

You must first create a results folder and an archives folder containing a folder for each taxid you want to study. For each taxid folders in results folder, you have to create fasta files named sequences_list.txt containing sequences you are looking for. For each taxid folders in archives folder, put your corresponding archives (fastq.gz files).

Load your TOP environment with conda

conda activate TOP_env

Launch your TOP pipeline

snakemake

Result/Output files

Structure of the output directory

├── Output
    ├── TaxId_1
    |	├── bbduk_log
    |	|	├── SampleId_1.log
    |	|	├── SampleId_2.log
    |	|	├── SampleId_....log        
    |	├── SampleId_1
    |	|	├──	rnaspades
    |	|	├── aln.sam
    |	|	├── aln_ref.sam
    |	|	├── CRISPR_contigs.fasta
    |	|	├── direction_result.txt
    |	|	├── rseqc_results.txt
    |	|	├── sens_archive.txt
    |	|	├── SampleId_1.csv
    |	|	├── SampleId_1.fq
    |	|	├── SampleId_1.txt
    |	|	├── SampleId_1_bbduk.fq
    |	├── SampleId_2
    |	├── SampleId...
    |	├── genomic.fna
    |	├── genomic.gff
    |	├── Results.csv
    |	├── sequences_list.txt
    |
    ├── TaxId_2
    ├── TaxId...

Description of result/output files

OutputFileName	Description
Results.csv	Contains all the results from all SampleID.csv from the TaxId directory.
SampleId.csv	Contains the final results summary of a sample. Output format is a csv with the following columns: Tax ID Archive's name Repeat sequence Number of CRISPR reads in archive % reads CRISPR in archive Number of reads in input sens % reads in input direction Number of reads in reverse input direction % reads in reverse input sens Repeat sens in archive p-value % Reads ++ in archive (RseqC) % Reads +- in archive (RseqC) Archive direction Repeat direction
genomic.fna	The assembly of reference genome or representative genome in fasta format.
genomic.gff	Annotation file of reference genome or representative genome in gff format.
CRISPR_contigs.fasta	Contigs build with rnaspades.py from bbduk.sk output sequence of interest and filtered by the script contigs_coverage.py

Description of scripts and TOP files

ScriptName	Description
aln.sh	Aligns reads of interest with contigs of interest and then filters that do not match.
assembly.csv	Table contains metadatas from RefSeq assemblies.
board.py	Builds the final results table.
contigs.sh	Create an empty assemly rnaspades file if rnaspades failed.
contigs_coverage.py	Filters assembly contigs by coverage rnaspades results.
fai.sh	Index contigs of sequences of interest.
full_link_fna.awk	Builds the full link to genome reference file.
full_link_gff.awk	Builds the full link to the annotation associated with reference genome.
link.py	Builds link to genome reference file.
orientation.py	Orientation results of the sequence of interest.
rnaspades.sh	Launches rnaspades.py to build contigs of sequence of interest. Creates empty file if there is an empty file in input.
RseqC.py	Filters results from RseqC
rseqc.sh	Launches infer_experiment.py from RseqC to orientate archive
sequences_list_format.py	Reformat sequence of interest in input (lower to uppercase ...)
Snakefile	File input used by snakemake
TOP.yaml	File used by conda to build the TOP environment