User:R. Eric Collins/GenomicsTutorial/Genomics/Mutant

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Contents

Overview

The purpose of this exercise is to become familiar with the following:

Concepts

  • Taxonomy
  • Shotgun sequencing

Techniques

  • Short read mapping
  • Visualization of reads

Software/Databases

  • NCBI Taxonomy (taxonomy database)
  • Galaxy (web server for genomics and metagenomics analysis)
  • Lastz (software for mapping small reads to a reference sequence)

Protocol

Get Genome Sequence
Mutate Sequence
  • go the Galaxy and log in if you have an account
  • go to Get Data --> Upload File
  • choose file 'sequence.fasta' that you just downloaded from NCBI
  • below under 'genome' select the genome you downloaded (this will tag the file for future reference)
  • click 'Execute' and wait for the upload to complete
  • click the pencil next to the uploaded file and change the name to something sensible
  • go to EMBOSS --> msbar and mutate at will
Simulate short read data
  • go to EMBOSS --> splitter
  • split msbar output at size <100 with short overlaps
Align short reads to Reference genome
  • go to NGS: Mapping --> Lastz
  • Align sequencing reads in: <splitter output>
  • Against reference sequences that are: <in your history>
  • Select a reference dataset: <your genome fasta file>
  • Select output format: <polymorphisms>
  • click Execute
  • wait to finish
  • click pencil icon next to output dataset
  • change Data Type --> bed
  • change Database/Build --> the genome you chose in NCBI
  • save the Attributes
Visualize Polymorphisms
  • click graph icon in output dataset = Visualize in Trackster
  • choose Insert into New Browser
  • for Reference Genome Build use the same as your genome
  • change "Select Chrom/Contig" to "chr"
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