Missing genes and did not produce circular mitogenome

[meeranhussain]

Hi,

I tried assembling the mitogenome of my insect species using MitoZ. The final assembly is not circular, with a size of 15,442 kb. Additionally, the following genes are missing:

l-rRNA
tRNA-Cys
tRNA-Glu
tRNA-Ser
tRNA-Val
I've tried different K-mers ranging from 57 to 129 and used SPAdes and MEGAHIT. However, the genome remains non-circular with the aforementioned missing genes.

Any suggestions to better use MitoZ to get a circular genome would be greatly appreciated.

Thank you!

[linzhi2013]

How about smaller kmers, e.g. 31, 35, 39, 45, 55?

[meeranhussain]

I did try small kmers with spades but still the same results. Also, FYI I tried mitogenome assembly using Flye on Oxford Nanopore Technology (ONT) long read data in meta mode and produced circularized genomes with sizes ranging from 29-32kb (had all the genes), which is unusually large for insect mitogenomes. I verified long-read mitogenome assembly method on Calliphora sp ONT data (whose mitogenome is typically 15-16kb). However, using Flye with this method resulted in a 32kb circular contig. which raises concerns about misassemblies by this method. Any suggestions you have would be helpful.
summary.txt

[linzhi2013]

You can annotate your mitogenome from ONT data with MitoZ and find out the breakpoint for a single circular mitogenome.

Or you can map the NGS reads to the ONT mitogenome and call consensus, and then also cut it based on gene annotation results.

[meeranhussain]

Thanks for your reply. I annotated ONT generated mitogenome using MitoZ which still didn't annotate following genes: "Potential missing genes:
#Gene total_missing_number
l-rRNA 1
tRNA-Cys 1
tRNA-Glu 1
tRNA-Ser 1
tRNA-Val 1"

But these genes were annotated using MITOS2 on ONT mitogenome, any reason for this difference?

[linzhi2013]

It could be that your sample is too divergent from the database MitoZ uses to annotate these genes.

[meeranhussain]

That makes sense. How to I find circular breakpoints in this Mitogenome, Is there any file generated during annotation?

[linzhi2013]

based on the coordinates of the repeated gene, such as COX1.