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

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)

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Protocol

Get Genome Sequence

• go to the NCBI Taxonomy Browser
• select your Domain of interest
• click "Display" after filtering for "has genome sequences"
• search or browse for a genome of interest
• click the name of the organism
• click on genome sequences on the right
• click on the RefSeq ID for the genome of interest
• click again on the ID
• http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Undef&id=2&lvl=3&p=mapview&p=has_linkout&p=blast_url&p=genome_blast&lin=f&keep=1&srchmode=1&unlock&filter=genome_filter
• click on FASTA
• click "Send" and choose Destination --> File and Format --> FASTA and click "Create File"
• file will be downloaded as sequence.fasta

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"