Hello group,
I have three samples that are confirmed by qPCR to be positive for our target virus(Cts between 17 and 23), the labs have been doing a few passages and have noticed an abnormal CPE indicating there is a possible contaminant, most likely considering these are tissue samples from the field. So we decided to go fishing and sequence the samples. We did two preps (DNA/RNA), and the RNA will be processed tomorrow. Now I'm trying to dig through the data.
My concern is when I use the target genome as a template I find it in the data, but the reads are very small. The mean mapping quality (according to QuailMap) is 3.25 across the genome. The depth of coverage is a mean of 180x. So it looks like it should be there but I was concerned since the reads that mapped are so small -- about 1/10th the expected size. So I used SPAdes to do a de novo assembly. In the assemblies I find a lot of the host, some weird unlikely hits (i.e. monkeys in china), but no target virus. So my conclusion is that in the sequencing run we did, we did not have the target virus in it.
I have a few ideas on why the discrepancy between the qPCR and the sequencing results, mostly making sure the same sample used for both. However I want to make sure that I'm not doing anything incorrectly from a bioinformatic standpoint. Is my conclusion supported by the results? Am I wrong in thinking that if I can do a reference assembly that I should be able to put together a piece in a de novo assembly, or is that a misconception?
