Gen2Epi

An automated whole-genome sequencing pipeline for linking full genomes to antimicrobial susceptibility and molecular epidemiological data in Neisseria gonorrhoeae.

Alternative links

https://github.com/DillonLab/Gen2Epi

ftp://www.cs.usask.ca/pub/combi

Requirements

1. Perl V5.16.3 or higher

2. Install the following programs and make them executable

    a.	FastQC 
    b.	MultiQC
    c.	Trimmomatic
    d.	Kraken
    e.	Bowtie2
    f.	SPAdes
    g.	BBMap
    h.	Ragout
    i.	Prodigal
    j.	QUAST
    k.	BLAST
    l.	EMBOSS
    m.	NGMASTER
   

3. If the installed programs are not executable, then all required softwares should be in path

a.	export PATH=$PATH:/installation-path/FastQC
b.	export PATH=$PATH:/installation-path/Trimmomatic-0.36
c.	export PATH=$PATH:/installation-path/SPAdes-3.11.1-Linux/bin
d.	export PATH=$PATH:/installation-path/SPAdes-3.11.1-Linux/share
e.	export PATH=$PATH:/installation-path/bbmap/
f.	export PATH=$PATH:/installation-path/ngmaster/
g.	export PATH=$PATH:/installation-path/x86_64
h.	export PATH=$PATH:/installation-path/ncbi-blast-2.6.0+/bin/
i.	export PATH=$PATH:/installation-path/EMBOSS-6.6.0/bin
j.	export PATH=$PATH:/installation-path/Sibelia-master
k.	export PATH=$PATH:/installation-path/Sibelia-master/bin
l.	export PATH=$PATH:/installation-path/Sibelia-master/bin/bin
m.	export PATH=$PATH:/installation-path/Sibelia-master/bin/lib
n.	export PATH=$PATH:/installation-path/Sibelia-master/bin/share
o.	export PATH=$PATH:/installation-path/fenderglass-Ragout-71562fc
p.	export PATH=$PATH:/installation-path/quast-4.5
q.	export PATH=$PATH:/installation-path/Prodigal-2.6.3
r.	export PATH=$PATH:/installation-path/bowtie2-2.3.2

Alternately, users can also copy the above-mentioned commands [a-r] in “.bashrc” and then set the path in the current 
working directory by running the following command

“source .bashrc” 

4. Open the terminal and use the following commands

    "git clone https://github.com/DillonLab/Gen2Epi.git"
   
    "cd Gen2Epi"
   

5. Copy the test dataset in the current working directory under

    “/home/user/Desktop/Test_DATA”
   

6. Copy the Gen2Epi_Scripts folder in your current working directory.

How to use

1. Prepare a tab-limited input file describing the full name and the paired-end read files, e.g.,

    WHO-F WHO-F_S2_L001_R1_001.fastq.gz WHO-F_S2_L001_R2_001.fastq.gz
    WHO-G WHO-G_S3_L001_R1_001.fastq.gz WHO-G_S3_L001_R2_001.fastq.gz
    WHO-K WHO-K_S4_L001_R1_001.fastq.gz WHO-K_S4_L001_R2_001.fastq.gz
    WHO-L WHO-L_S5_L001_R1_001.fastq.gz WHO-L_S5_L001_R2_001.fastq.gz
   

First column = Sample ID

Second Column = First fastq read pair

Third Column = Second fastq read pair

Note: Make sure to put all the fastq reads in the same folder.

If you have thousands of samples then the input file in the above-mentioned format can be prepared by using the following script:

    “perl Prepare_Input.pl <path-to-fastq-files> <number e.g 12>”

2. For Quality Check and Trimming

    "perl WGS_SIBP_P1.pl /home/user/Desktop/Test_DATA /home/user/Desktop/Test_DATA/WHO_Data both 3 3 4:15 30”
   

3. For De-novo assembly

    “perl WGS_SIBP_P2.pl /home/user/Desktop/Test_DATA/Input Trimming trimmed 2”
   

4. For Chromosome Scaffolding

    “Perl WGS_SIBP_P3-Chr-C1.pl /home/user/Desktop/Test_DATA/Input /home/user/Desktop/Test_DATA/WHO_Full_Reference_genome/Chromosome /home/user/Desktop/Gen2Epi_Scripts/Chrom_AssemblyTrimmedReads /home/user/Desktop/Test_DATA/WHO_Genome_Annotation/Chromosome 1 TXT”
   

5. For Plasmid-type identification

    “Perl WGS_SIBP_P3-Plas_C1.pl /home/user/Desktop/Test_DATA/Input Plasmid_AssemblyTrimmedReads 1 /home/user/Desktop/Test_DATA/Plasmid.fasta”
   

6. For Epidemiological analysis and AMR prediction of the assembled scaffolds: Please make sure to delete the existing output file before running the following commands.

   a. NG-MAST

    “perl WGS_SIBP_P4_Epi.pl /home/user/Desktop/Test_DATA/Input Chr_Scaffolds NGMAST”
   

   b. NG-MAST

    “perl WGS_SIBP_P4_Epi.pl /home/user/Desktop/Test_DATA/Input Chr_Scaffolds MLST MLST-Genes.fasta MLST_alleles.fasta pubMLST_profile.txt”
   
    Please Note: In case you encounter “BLAST database index” error then make sure to build the blast database for “MLST-Genes.fasta” using the following command:
    “makeblastdb –in MLST-Genes.fasta –db nucl”
   

   c. NG-MAST

    “perl WGS_SIBP_P4_Epi.pl /home/user/Desktop/Test_DATA/Input Chr_Scaffolds ngstar AMR-Genes-NgStar.fasta AMR-Genes-NgStar-alleles.fasta”
   

   d. Chromosome-mediated Tetracycline Resistance

    “perl TetRes.pl rpsJ.fasta Chr_Scaffolds/All_Sequences TetResOut”
    “perl SeqProt.pl TetResOut”
   

Contacts

Dr. Reema Singh: [email protected] ; [email protected]

Citation

Singh R, Dillon JA, Demczuk W, Kusalik A. Gen2Epi: an automated whole-genome sequencing pipeline for linking full genomes to antimicrobial susceptibility and molecular epidemiological data in Neisseria gonorrhoeae. BMC genomics 2019;20(1):1-1.