Building Initial Alignment
OverviewTeaching: 0 min
Exercises: 0 minQuestions
Key question (FIXME)Objectives
First learning objective. (FIXME)
Assemble a set of close outgroup sequences
Outgroup sequences must be full-length. You should include outgroups from all of the clades that are neighbors to your group of interest, 5-10 sequences per group. These should be chosen based on your knowledge (ask for help if needed). You can get outgroup sequences from GenBank, SILVA or PR2. See step 2 for details. Add your outgroup sequences to clustered starting sequences (NAME.clustered.fasta file)
Align using MAFFT.
Use the default MAFFT algorithm (command below) unless there is a specific reason to use a different algorithm or approach. (Experience users can use different preferred alignment program)
mafft --reorder --auto NAME.clustered.fasta > NAME_aligned.fasta
Alternative: Align using INFERNAL
cmalign --outformat AFA -o NAME_aligned_infernal.fasta SSUref.cmm NAME.clustered.fasta sed 's/\./-/g' NAME_aligned_infernal.fasta > NAME_aligned.fasta
Alternative: Align using SINA*
./sina -i NAME.clustered.fasta -o NAME_aligned.fasta --outtype fasta --ptdb REFERENCE_ALN.ARB
If you choose to use SINA alignment, that you have to provide a reference alignment in ARB format. Easiest way to get one is to downloaded from SILVA’s ARB database page.
Trim the alignment using trimal.
trimal -in NAME_aligned.fasta -out NAME.trimal.fasta -gt 0.3 -st 0.001
(NOTE: Add explanation of options and flags for all function calls.)
First key point. Brief Answer to questions. (FIXME)