Detection of polymorphisms from yeast NGS by PED

• 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.

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=ERR4194721
  $ perl download.pl accession=ERR4194722
  $ perl download.pl accession=ERR4194723
  $ perl download.pl accession=ERR4194724
  $ perl download.pl accession=ERR4194725
  $ perl download.pl accession=ERR4194726
  

  Scripts are updated by git pull command.
  Sometimes error message will appear. The script will try to reconnect. Check increasing size of downloading files.
  
• Detection of polymorphisms in NGS sequences on yeast reference genome sacCer3.

  $ perl ped.pl target=ERR4194721,control=ERR4194722,ref=sacCer3
  $ perl ped.pl target=ERR4194722,control=ERR4194723,ref=sacCer3
  $ perl ped.pl target=ERR4194723,control=ERR4194724,ref=sacCer3
  $ perl ped.pl target=ERR4194724,control=ERR4194725,ref=sacCer3
  $ perl ped.pl target=ERR4194725,control=ERR4194726,ref=sacCer3
  

  Since sacCer3 reference file is already configured, data are automatically downloaded and calculated.
  When all processes are done, vcf files will be created in target derectory.
  Other strain belongs same project (condition) should be 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

• Set up 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=ERR4194721,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl download.pl accession=ERR4194722,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl download.pl accession=ERR4194723,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl download.pl accession=ERR4194724,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl download.pl accession=ERR4194725,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl download.pl accession=ERR4194726,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl ped.pl target=ERR4194721,control=ERR4194722,ref=sacCer3,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl ped.pl target=ERR4194722,control=ERR4194723,ref=sacCer3,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl ped.pl target=ERR4194723,control=ERR4194724,ref=sacCer3,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl ped.pl target=ERR4194724,control=ERR4194725,ref=sacCer3,wd=/work
  $ sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl ped.pl target=ERR4194725,control=ERR4194726,ref=sacCer3,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 pre-analyzed vcf files linked below.
  • Import files by selecting 'Local file' in 'Tracks' pull down menu.
    
• Or download from http://software.broadinstitute.org/software/igv/download.
  Drag vcf files on main display panel of IVG, data will be imported.
  When click the number of chromosome, map and polymorphisms by each chromosome will be shown.
  Click at interested position, zoom up the region.
  Mouse over at vertical line of polymorphism, detailed information will be shown.
  Sequence of primer pair for target polymorphism and PCR product size as well as kind and frequency of polymorphism will also be shown.
  
• ERR4194721.vcf
  ERR4194722.vcf
  ERR4194723.vcf
  are already analyzed data.

Link of PED

• Github: https://github.com/akiomiyao/ped/
• Docker: sudo docker pull akiomiyao/ped
      sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl download.pl accession=ERR194146,wd=/work
      sudo docker run -w /ped -v `pwd`:/work akiomiyao/ped perl ped.pl target=ERR194146,ref=hg19,wd=/work
  
• Paper: Polymorphic edge detection (PED): two efficient methods of polymorphism detection from next-generation sequencing data (2019) BMC Bioinformatics 20(1):362

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