We have sequenced two plasmids with a Novaseq machine (reads length of 100 bp, paired end).
For one of them, we have aligned the reads against the reference genome using BWA-mem and, as the reads mapped across the full length of the reference genome, we have extracted the sequence of the plasmid on IGV (with the feature extract consensus sequence):
Therefore, it would be meaningless to extract the consensus sequence from IGV. We then sought to perform denovo assembly using Spades. We ran the following command
The thing is that Spades outputs, among others, a scaffold.fasta and a contigs,fasta file. I was now wondering how (if) is it possible to get the continuous plasmid sequence from the Spades output?
Please see How to add images to a Biostars post to add your images properly. You need the direct link to the image, not the link to the webpage that has the image embedded (which is what you have used here)
The sequence of both contigs.fasta and scaffolds.fasta are nearly identical, the difference being the scaffolds.fasta possibly contains some contigs merged due to paired read mapping information. These scaffolds would contain some Ns indicating where is the gap in the sequence.
how (if) is it possible to get the continuous plasmid sequence from the Spades output?
The if depends on SPAdes assembling the plasmid into one continuous contig. There are several options as to how, one being:
make a blast database with the contigs.fasta (and / or scaffolds.fasta),
blast the reference plasmid against the database prepared in the previous step,
That's useful, though as the length of the scaffolds are shorter than the full sequence of the plasmid (obviously) I can only check whether the scaffolds sequence are correct. However the contiguous sequence i.e. how the scaffolds are connected to one another is still missing.
I didn't state clearly, but I think the second plasmid is very different from the first and from the reference. The mapping suggests there are several deletions on the second plasmid compared to the first.
Assembling Illumina data will generally result in a lot more contigs than originally expected, one of the reasons is sequencing errors and contaminants. These can be filtered out by looking at the coverage, as the "proper" contigs will have a higher coverage than the garbage contigs.
We have checked the sequencing parameters and they all look OK. So I am more tempted to believe that the sequenced of the sequenced plasmid is not the same as the reference.
Please see How to add images to a Biostars post to add your images properly. You need the direct link to the image, not the link to the webpage that has the image embedded (which is what you have used here)