Evidences of circularization could not be found, but everyother step was successful

[eugeniosss]

Hello,
I have been trying to assemble a full mitogenome with MitoFinder using megahit. I have been able to assemble a contig of ~16,500 bp, which should be quite close to the size of the genome. However, it seems that I am falling short in the last step, as it does not find "Evidences of circularization"... Could you give me a hint or recommendation of how I should proced to fully generate a whole mitogenome that finds circularization? Please bear in mind I do not have a available reference genome for the species I am dealing with. Some details regarding the input: They are 33 million paired end reads with an insert size ~350bp, which were trimmed and quality filtered with AdapterRemoval. I also leave here the log file.

PRN_megahit_MitoFinder.log

[RemiAllio]

Hi!

Indeed, the mitogenome is close to being complete if not yet complete.
There are two things: either megahit failed in assembling a part of the genome (most likely the D-loop) or MitoFinder fails to find the circularization while the mitogenome is full (I had a report of this recently and unfortunately didn't have time yet to go back to the code to check).

There are several things you can do. The first one is to use MetaSPAdes instead of megahit and see if the results are similar.
The second is to use a mapping-based strategy to try to assemble the rest of the mitogenome and give the result back to MitoFinder for annotation and circularization.

In the meantime, I will try to go back to my code to see if something is wrong.

Best,
Rémi

[eugeniosss]

I already tried to run with MetaSPAdes, unfortunately I have not been very successfull... It always gives me an error, either -6 or -9... I attach one of the logs of my attemtps.
metaspades.log
I know this is a completely different error, but do you have any idea of what may be causing it?

[RemiAllio]

Hi,

Did you try to run MitoFinder using its singularity container?

Otherwise, it seems that the -6 -9 errors can be associated with memory issues. How much RAM do you have?

Best,
Rémi

[eugeniosss]

Hello,
Thank you very much for your quick replies!
I tried to install with singularity, but I am running Mitofinder on a server, where I do not have sudo privileges. Therefore, I am not certain if is possible to run Mitofinder with singularity (?)
I have 104789 MB of RAM and I am trying to do a run now with -m 100000, should be enough, right?

[RemiAllio]

Hello,
Yes, this should be more than enough... If it doesn't work, it means that the problem comes from your system configuration and you will have to discuss it with your cluster administrator.

Indeed, to be able to use the container you need to have singularity v >=3.0 installed on the cluster or ask an administrator to install it. That the only thing you need. So, once singularity is available, you can run MitoFinder without privileges.