I am having some difficulties assembling a mitogenome. These are the steps I followed:
- I sequenced the total DNA from a leech bacteriome using Illumina Hiseq X (150 bp paired-end reads).
- After quality control and adapter trimming, the metagenome was assembled using SPAdes 13.3.0
- I did run locally blastx in order to identify which scaffolds belong to leech mitochondrial genome resulting ~89 scaffolds matched with one closely related mitogenome from a closely related leech species (I built the reference database using some leech genomes, one genome of an endosymbiotic bacteria of the leech related species and its mitogenome).
- These 89 scaffolds were mapped versus the reference mitogenome employed in the previous step using Mummer (Promer), in order to find which scaffolds align with the reference mitogenome. It resulted in ~18 scaffolds
- Once separated the above mentioned scaffolds, I choosed those scaffolds with a length below 15 Kb ( the average mitogenome length for leeches) and with high coverage values (preferably those with values > 100x).
- I did a blastx online for each of the selected scaffolds in an effort to identify what genes were present in each sequence by finding conserved domains and discard regions with compositionally biased regions and non mitochondrial DNA regions.
- With previous knowledge of mitogenome sinteny, I aligned the scaffolds containing contiguous genes with the aim of determining overlapping regions. Then, the scaffolds were edited to merge them.
- Once merged the scaffolds, I annotated the sequence using MITOS2 web server and apparently I got the complete mitogenome.
I obtained a SAM file of the assembly which was visualized in Tablet and I noticed some conflicting regions suggesting a chimeric assembly.
I wonder how I could deal with this chimeric assembly. Can someone suggest me a strategy to do the assembly in order to avoid obtaining chimeras? Is there any other strategy to select and merge the scaffolds?
Thanks in advance!!!!