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Old 07-04-2014, 01:57 AM   #1
francicco
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Default mtDNA de-novo assembly

Dear assemblers,

I'm trying to assemble the complete mtDNA genome of a Drosophila species using pair-ends transcriptome reads. To do so I'm using MIRA 4 with MITObim 1.7 following the protocol described here (https://github.com/chrishah/MITObim).
As first step I aligned all reads with bowtie to all available complete mtDNAs from Drosophila species and I took the highest mapped mt as the backbone for MIRA as considered the most closely related having higher fraction of conserved regions.

I start from 140M reads in paired-ends (140M+140M) too much. MIRA give me clear warning, too much coverage. So I'm splitting reads in blocks in order to get a better coverage. I have few questions, maybe you could help me.

- What is the best coverage I should search for?
- Does It make sense to perform x reconstruction, each of them with different subsets of reads and take the consensus of the alined de-novo genome?
- From a preliminary analysis I used the 50% of reads. MITObin starts the iterative process and it stops at the iteration #2. Then I aligned iteration #1, #2 with the backbone and other 2 complete mtDNAs. The iteration #2 looks much worst reconstructed than #1, with a lot on long gaps. Is it normal and should I take it anyway as best de-novo assembly?
- How important is the backbone, how much is its bias in the genome reconstruction?

Thank you so much for your help
Francesco
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Old 07-07-2014, 04:24 AM   #2
TiborNagy
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For bacteria 25x coverage is good.
Backbone is very important. Mira can handle SNP and short indels (4-5 nt length), but you won't see longer variations.
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