3. Generating a Consensus Sequence

3.1 Mapping trimmed reads to reference

Note

This is optional if the reference for the primer design and the preferred reference for consensus generation are different. Otherwise simply use the clipped mapping file from the previous step.

Similar to what we did before we now map the trimmed reads to our preferred reference genome.

minimap2 -Y -t {threads} -x map-ont -a {reference} {input} | \
samtools view -bF 4 - | samtools sort -@ {threads} - > {output}
  • {reference} is the fasta file containing the preferred reference.
  • {input} is the trimmed fastq file from step 2.4.
  • {output} is the mapping file, it could be named something like barcode01_mapped.bam

3.2 Creating consensus from filtered mutations

Virconsens is a tool written by David to create a consensus sequence from Nanopore amplicon reads mapped to a reference in a mapping .bam file.

It works by reading the mapping file position-by-position and counting the mutations, insertions and deletions. The mutation or deletion (or original nucleotide from the reference) with the highest count is considered the “consensus” at that position.

In the next step it filters positions with too low coverage based on the mindepth threshold or too low frequency based on the minAF threshold.

The last step of the tool is to iterate over the reference genome and replace the reference nucleotide with the mutation, insertion or deletion, replace filtered position with “N”, or keep the original reference nucleotide. (1 and 2 nucleotide indels are ignored as they are very often erroneous).

Before running virconsens we have to index the .bam mapping file.

samtools index {input}

virconsens \
-b {input} \
-o {output.fa} \
-vf {output.vf} \
-n {name} \
-r {reference} \
-d {coverage} \
-af 0.1 \
-c {threads}
  • {input} is the mapping bam file from step 3.1.
  • {output.fa} is the fasta file containing the consensus sequence (e.g. barcode01_consensus.fasta)
  • {output.vf} is an optional tsv file which contains variant information.
  • {name} is the custom name of your sequence that will be used in the fasta file (e.g. barcode01_consensus)
  • {reference} is the fasta file containing the preferred reference, the same as in the previous step.
  • {coverage} is the minimal depth at which to not consider any alternative alleles.
Note

We now have a consensus sequence of our sequencing result. This is the “raw” result we can continue using for multiple sequence alignment and phylogeny in the next chapter.