Hello, how are you?
I'm performing an analysis using Illumina sequences prepared with a library ribo-zero detector. These are insect and bat samples that tested positive for some arbovirus via RT-PCR.
I'd like to know how to identify which viruses these are, since many have a negative-sense RNA (meaning, to be transcribed in the cell, they need to be transcribed by the associated polymerase from 3'-5' to 5'-3').
I've tried several approaches: I mapped the fastq against the host genome and retrieved the unmapped reads, assembled them with MEGAHIT, and classified them with Kraken, but it didn't work. I did the same thing, but mapping with the viral genomes and retrieving the mapped reads, and it also didn't work.
I've used seqkit -r to reverse-sequence the sequences and achieve the same results, but nothing. Does anyone have any suggestions for a pipeline/tool that could help me, please?
Thank you for your attention and even more for your response!
Hugs
You can use Kraken2 to directly classify the reads.
Tutorial: https://cloud-span.github.io/nerc-metagenomics06-taxonomic-anno/01-taxonomic/index.html
Databases: Kraken2 Metagenomic Virus Database, MinusB
Hi, thanks for answer!
I'm using Kraken2, but the classified reads, when put on BLAST, didn't returned nothing, even in Viruses_db...
It's possible/likely that your sequencing simply didn't recover any virus reads, despite RT-PCR detection. Often there is too much 'other stuff' in samples, e.g. host nucleic acids or bacteria, meaning that the viruses aren't sampled. Without a more complete description of how you performed the sequencing it's difficult to say for sure.