Detection of polymorphisms from Bacillus subtilis NGS by PED

[Link to top page]

In this section, install of Linux(Ubuntu), setup of PED and browsing of detected polymorphisms using NGS of mutant strains of Bacillus subtilis 168 are described.

An example of genome view by IGV.Click to large.
Indels can be detect exactly, as well as SNPs.
Nucleotide sequences of primer pair to amplify target mutations are also shown.

Strain GP1948 (SRR7967955) is a suppressor mutant of topoisomerase A mutation.
Mutation at 2,011,091 is G to A substitution in the GltA gene.
Reference: Topoisomerase IV can functionally replace all type 1A topoisomerases in Bacillus subtilis

Install of Ubuntu

Download Ubuntu 20.4 LTS from Ubuntu download site.
Ubuntu install tutorial will be helpful for installing.
Because PED uses maximum CPU power, exclusive use for PED analysis is recommended.
PED supports multithread environment. CPU with 4 or 8 core is recommended.

Setup and analysis of PED

Installing software required by PED

$ sudo apt update
$ sudo apt upgrade
$ sudo apt install curl
$ sudo apt install git
$ git clone https://github.com/akiomiyao/ped.git

To download sequence data, fastq-dump from NCBI is required.
Tool kit can be download from https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software
Details of setup fastq-dump is described in https://akiomiyao.github.io/ped/sratoolkit/index.html
Download fastq files from NCBI Sequence read archive site.
```
$ cd ped
$ git pull
$ perl download.pl accession=SRR7967955
```
Scripts are updated by git pull command.
Sometimes error message will appear during download. The script will try to reconnect. Check increasing size of downloading files.
Check sequence length of the fastq file.
```
$ perl check_length.pl SRR7967955
Omitted above
290     4868
291     4953
292     3574
293     2917
294     4086
295     2267
296     5828
297     9657
298     32047
299     80536
300     536716
301     1153100
```
Because PED requires fixed length short read sequences, clipping option is required.
Longest sequences (301 base) are most abundant. But, there are many shorter sequences in the file.
In this case, 150 base for clipping is better. Long sequence with 300 and 301 base will divided into 2 sequences.
Detection of polymorphisms in NGS sequences on Bacillus subtilis reference genome.
```
$ perl ped.pl target=SRR7967955,ref=Bsubtilis,clipping=150
```
Since Bsubtilis reference file is already configured, data are automatically downloaded and calculated.
When all processes are done, vcf files will be created in target directory.
If sequence from other strain belongs same project (condition) is chosen for the control, common mutations among strains against the reference sequence will be eliminated.
Sequences of primer pair are also outputted into vcf file.

For Docker users

Install of Docker

$ sudo apt install docker
$ sudo apt install docker.io

Example of analysis,

$ sudo docker pull akiomiyao/ped
$ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl download.pl accession=SRR7967955,wd=/work
$ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl check_length.pl accession=SRR7967955,wd=/work
$ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl ped.pl target=SRR7967955,ref=Bsubtilis,clipping=150,wd=/work

Copy and paste commands described above to your terminal window, change values of accession, target and clipping, and run.
Both the ped.pl script and the docker container can be run on Google Cloud Platform and Amazon Web Services.

Visualization of detected polymorphisms using IVG

Integrative Genomics Viewer (IVG) is a genome browser made in Broad Institute.
Web application of IVG https://igv.org/app/ is useful.
Click to large.
- Download fasta file of reference genome Bsubtilis.fasta and the index file Bsubtilis.fasta.fai.
- Select 'Local File ...' in pull down menu of 'Genome' and submit both Bsubtilis.fasta and Bsubtilis.fasta.fai files.
- Download gff file. GCF_000009045.1_ASM904v1_genomic.gff.gz
- Select 'Local File ...' in pull down menu of 'Tracks' and submit GCF_000009045.1_ASM904v1_genomic.gff.gz file.
- Download pre-analyzed vcf file SRR7967955.vcf.
- Import files by selecting 'Local file' in 'Tracks' pull down menu.
- Click '+' to zoom up. Vertical lines of mutations will be shown.
Or download from http://software.broadinstitute.org/software/igv/download.
Drag vcf files on main display panel of IVG, data will be imported.
Click at interested position, zoom up the region.
Mouse over at vertical line of polymorphism, detailed information will be shown.

Link of PED

Github: https://github.com/akiomiyao/ped/
Docker: sudo docker pull akiomiyao/ped
Paper: Polymorphic edge detection (PED): two efficient methods of polymorphism detection from next-generation sequencing data (2019) BMC Bioinformatics 20(1):362

Contact

MIYAO Akio, Ph.D (miyao@affrc.go.jp) (Family name is MIYAO)
Institute of Crop Science / NARO
2-1-2, Kannondai, Tsukuba, Ibaraki, 305-8518, Japan