First steps

1. Log in into the server
2. You should find a directory called sequences in your home.
3. List the files in that directory (for example with ls -l ~/sequences)
4. The files are compressed, we will try not to decompress them as it’s a good practice in metagenomics (to save lots of disk space and time)
5. To count the sequences in FASTA files, we can use grep or dedicated tools.
   • Try with grep first (for example with zcat FILE | grep -c ‘>’, or cat FILE | gzip -d | grep -c \> if zcat is not available)
   • We have SeqFu installed in our machine, so we can test seqfu stats --nice sequences/*gz.

A finished bacterial genome

1. The file GCF_000027325.fasta.gz contains a finished bacterial genome, as we saw from the stats it’s a single sequence.
2. Print the name of the first sequence,for example with zcat ./sequences/GCF_000027325.fasta.gz | grep \>
3. (Optional) Now print the first lines of the file, and copy a small portion (~200 nucleotides) in a new file in FASTA format, calling it ~/sequences/myco-bit.fa, and calling the sequence itself Seq1.
   • You can use the nano editor - that is widely available in most servers - or an improved alternative called micro.

Making symbolic links

• Sometimes it’s useful to make in our favourite locations a link to files existing elsewhere. Remember that the link will break if we move/remove the source file. We have an Illumina paired end sample stored in /data/cami/simple_R1.fq.gz (and its corresponding R2). The command to make symbolic links is ln -s SOURCE DESTINATION.

Try:

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ln -s /data/cami/simple_* ~/sequences/

• With ls -l, note how symbolic links are rendered.
• SeqFu has tools resembling the GNU commands but for sequences, like seqfu head, seqfu tail, seqfu grep, plus other utilities. Try seqfu head.
• Count the reads present in the files,for example with seqfu count sequences/simple*
  • This command can take some time, why don’t we save its output to a file instead: seqfu count sequences/simple_* > sequences/simple_counts.txt & (the final & will send the process to the background)
• We now want to make a subsample. To do this we can use seqfu head --skip 10 FILE > SUBSAMPLED. Let’s try a “for loop”:

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mkdir subsampled

for FQFILE in sequences/simple_R*; 
do 
   echo $i; 
   seqfu head --skip 10 $FQFILE > subsampled/$(basename $FQFILE); 
done