Bacterial genome assembly

Fall 2014 BBB talk

Raj Ayyampalayam

Institute of Bioinformatics

Quantitative Biology Consulting Group

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Bacterial genomes

• Less complicated than larger genomes
• Has the same issues
• Garbage in garbage out
  • Better DNA better sequences
  • PCR free library perp
  • Multiple DNA extraction (different techniques)
• Size ranges from 2MB to 5MB

Sequencing technologies

• Illumina
  • MiSeq 
    • PE 150 - 4.5 GB to 5 GB
      • ~ 20 Genomes in a run
    • PE 300 - 13 GB to 15 GB
      • ~ 40 - 50 Genomes in a run
  • NextSeq
    • PE 150
      • Med output kit
        • 30 GB per run 
          • 50 - 60 Genomes
      • High output kit
        • 100 GB per run
          • 150 Genomes
      • Spike-ins?

Sequencing technology cont...

• Long reads
  • PacBio
    • ~ 300 MB to 500 MB of data per SMRTCell
    • Reads of length up 30 KB
• Sequencing strategy
  • All Illumina
    • Single library
    • Multiple library
  • Illumina and PacBio
    • Single Illumina fragment library (50x)
    • PacBio library (20x)

Assembly programs

• A5 pipeline (http://sourceforge.net/projects/ngopt/)
  • ​Short reads only assembler
• SPAdes (http://bioinf.spbau.ru/spades)
  • ​Can use PacBio sub-reads for scaffolding and repeat resolution
• MaSuRCA (http://www.genome.umd.edu/masurca.html)
  • ​Hybrid assembler
  • Can user long reads for assembly
• PBcR (http://wgs-assembler.sourceforge.net/wiki/index.php/PacBioToCA)
  • ​Pacbio specific de novo assembler
  • With enough coverage can self correct reads
  • Can use Illumina reads to correct PacBio reads and then assemble the corrected reads

• PBJelly (http://sourceforge.net/p/pb-jelly/wiki/Home/)

  • Scaffolds draft assembly using long reads

Assembly metrics

• QUAST: Quality Assessment Tool for Genome Assemblies
  • http://bioinf.spbau.ru/quast
  • Allows for comparison of multiple assemblies at the same time
  • Graphical outputs 
  • Generates “predicted genes” metrics and coordinates 
• PHAST: PHAge Seach Tool
  • http://phast.wishartlab.com/
• Summarize Assembly
  • Part of PBJelly suite

Genome Assemblers Comparision

• GAGE-B http://bioinformatics.oxfordjournals.org/content/29/14/1718.full
  • ​MaSuRCA Best?
• SPAdes analysis using the new version
  • http://bioinf.spbau.ru/en/content/spades-30-gage-b-data-sets
• My experience
  • Depends on the nature of the genome
  • Try different assemblies
  • Tune and tweak parameters
  • Make your own meta assembly
    • http://garm-meta-assem.sourceforge.net/

Questions?Comments