Retrieve an Initial Set of Sequences and Cluster
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Exercises: 0 minQuestions
Key question (FIXME)Objectives
First learning objective. (FIXME)
Users can choose a different similarity threshold if desired. For example, you may wish to work at 99% identity for small clades. In this case, replace 0.97 with 0.99 throughout.
In all commands, you must substitute your clade name for “NAME”.
Retrieve initial sequences
We offer three ways of doing this - using GenBank, PR2 or SILVA. We recommend using SILVA or PR2 database. Use the grep command to pull out your sequences of interest. Note that clade names often differ in these different databases.
grep –A 1 -i “NAME” silva_reference_database.fasta > NAME.fasta
This will target targeted cultured isolates or isolates that were morphologically identified After you have downloaded your sequences, clean fasta headers to contain the GenBank accession number only.
sed 's/gi\|[0-9]*\|gb\|//' NAMEgb.fasta | sed 's/\..*//' > NAME.fasta
Cluster your sequences
Clustering will allow you to produce a manageable number of sequences using usearch (two commands). Similarity threshold is set at 97% here but can be adjusted by changing the “-id” command. These commands choose the longest sequence as the representative sequence for each cluster. We will use also this step to remove sequences shorter than 500 bp. You will use these representative sequences for your initial alignment/tree.
usearch -sortbylength NAME.fasta -fastaout NAME.sorted.fasta -minseqlength 500 -notrunclabels usearch -cluster_smallmem NAME.sorted.fasta -id 0.97 -centroids NAME.clustered.fasta -uc NAME.cluste
First key point. Brief Answer to questions. (FIXME)