Sequence Alignment for Phylogenetic Analysis: Difference between revisions
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== Locate Sequences and Generate FASTA File == | == Locate Sequences and Generate FASTA File == | ||
* The easiest way to find sequences is to start with a seed sequence then do BLAST searches restricting to RefSeq and the species of interest. | |||
* To find a seed sequence start with NCBI Gene, then find the first Refseq mRNA (should start with NM) then click on that and find the protein (should start with NP) | |||
* Paste that into your FASTA file (see next section) and name accordingly. | |||
* Paste that sequence or its NP id into [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome NCBI Protein Blast]. | |||
* Set the parameters to: | |||
** Database: Reference Proteins (refseq_protein) | |||
** Organism: Start with mouse (''Mus musculus'') or human (''Homo sapiens''), depending on your goal consider adding zebrafish (''Danio rerio''), ''Drosophila melanogaster'', chicken (''Gallus gallus'') and ''Caenorhabditis elegans'' | |||
=== Generating a FASTA File=== | |||
* FASTA format is described [https://zhanglab.ccmb.med.umich.edu/FASTA/ here], and [https://en.wikipedia.org/wiki/FASTA_format here] you need each sequence to start with a >SEQUENCENAME followed by a return and then the sequence, in this case the protein sequence. An example of a FASTA file would be: | |||
<code> | |||
>SEQUENCE_1 | |||
MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG | |||
LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK | |||
IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL | |||
MGQFYVMDDKKTVEQVIAEKEKEFGGKIKIVEFICFEVGEGLEKKTEDFAAEVAAQL | |||
>SEQUENCE_2 | |||
SATVSEINSETDFVAKNDQFIALTKDTTAHIQSNSLQSVEELHSSTINGVKFEEYLKSQI | |||
ATIGENLVVRRFATLKAGANGVVNGYIHTNGRVGVVIAAACDSAEVASKSRDLLRQICMH | |||
</code> | |||
* Save sequences in notepad, [https://notepad-plus-plus.org/ notepad++] or [https://www.sublimetext.com/ sublime] (not Word) as a <FILENAME>.fasta file. | |||
* Sequence names cannot have spaces. Generally its better to name it as '''mm_Gdf15-NM_004864.4''' where mm indicates mouse, Gdf15 is the gene name and NM indicates a [https://www.ncbi.nlm.nih.gov/refseq/ RefSeq mRNA]. If there are multiple mRNA's for the gene, name them | |||
== Create Multiple Sequence Alignment using CLUSTAL Omega == | == Create Multiple Sequence Alignment using CLUSTAL Omega == | ||
* CLUSTAL Omega is available at https://www.ebi.ac.uk/Tools/msa/clustalo/ | * CLUSTAL Omega is available at https://www.ebi.ac.uk/Tools/msa/clustalo/ | ||
* Select output format NEXUS to import into Mr Bayes | * Select output format NEXUS to import into Mr Bayes or PHYLIP format to import into PhyoBayes | ||
* Generate phlogenetic trees with Mr. Bayes [[Using Mr Bayes to For Phlyogenetic Analysis]] | * Generate phlogenetic trees with [http://megasun.bch.umontreal.ca/People/lartillot/www/download.html PhyloBayes] or Mr. Bayes [[Using Mr Bayes to For Phlyogenetic Analysis]]. | ||
=== PhyloBayes Analysis === | |||
* Mark in your notes the software version used. | |||
* The PhyloBayes manual can be found [http://megasun.bch.umontreal.ca/People/lartillot/www/phylobayes4.1.pdf here]. | |||
Latest revision as of 13:16, 18 April 2019
Locate Sequences and Generate FASTA File
- The easiest way to find sequences is to start with a seed sequence then do BLAST searches restricting to RefSeq and the species of interest.
- To find a seed sequence start with NCBI Gene, then find the first Refseq mRNA (should start with NM) then click on that and find the protein (should start with NP)
- Paste that into your FASTA file (see next section) and name accordingly.
- Paste that sequence or its NP id into NCBI Protein Blast.
- Set the parameters to:
- Database: Reference Proteins (refseq_protein)
- Organism: Start with mouse (Mus musculus) or human (Homo sapiens), depending on your goal consider adding zebrafish (Danio rerio), Drosophila melanogaster, chicken (Gallus gallus) and Caenorhabditis elegans
Generating a FASTA File
- FASTA format is described here, and here you need each sequence to start with a >SEQUENCENAME followed by a return and then the sequence, in this case the protein sequence. An example of a FASTA file would be:
>SEQUENCE_1
MTEITAAMVKELRESTGAGMMDCKNALSETNGDFDKAVQLLREKGLGKAAKKADRLAAEG
LVSVKVSDDFTIAAMRPSYLSYEDLDMTFVENEYKALVAELEKENEERRRLKDPNKPEHK
IPQFASRKQLSDAILKEAEEKIKEELKAQGKPEKIWDNIIPGKMNSFIADNSQLDSKLTL
MGQFYVMDDKKTVEQVIAEKEKEFGGKIKIVEFICFEVGEGLEKKTEDFAAEVAAQL
>SEQUENCE_2
SATVSEINSETDFVAKNDQFIALTKDTTAHIQSNSLQSVEELHSSTINGVKFEEYLKSQI
ATIGENLVVRRFATLKAGANGVVNGYIHTNGRVGVVIAAACDSAEVASKSRDLLRQICMH
- Save sequences in notepad, notepad++ or sublime (not Word) as a <FILENAME>.fasta file.
- Sequence names cannot have spaces. Generally its better to name it as mm_Gdf15-NM_004864.4 where mm indicates mouse, Gdf15 is the gene name and NM indicates a RefSeq mRNA. If there are multiple mRNA's for the gene, name them
Create Multiple Sequence Alignment using CLUSTAL Omega
- CLUSTAL Omega is available at https://www.ebi.ac.uk/Tools/msa/clustalo/
- Select output format NEXUS to import into Mr Bayes or PHYLIP format to import into PhyoBayes
- Generate phlogenetic trees with PhyloBayes or Mr. Bayes Using Mr Bayes to For Phlyogenetic Analysis.
PhyloBayes Analysis
- Mark in your notes the software version used.
- The PhyloBayes manual can be found here.
