Building Initial Alignment

Overview

Teaching: 0 min
Exercises: 0 min

Questions

• 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.)

Key Points

• First key point. Brief Answer to questions. (FIXME)