Sequence Alignment for Phylogenetic Analysis

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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

PhyloBayes Analysis

  • Mark in your notes the software version used.
  • The PhyloBayes manual can be found here.