Comparative Genome Analysis of

Question

[JJ13]

Hello.

I am currently working on a laboratory evolution project. I have a background in molecular biology and are a bit lost with the bioinformatics.

My project involves the culturing of a specific bacterial isolate under different stress conditions for several generations. This was done for 3 different conditions in triplicate, after which the DNA (as well as that of wild-type/reference) was extracted and send for full genome sequencing. The genomes were sequenced on the llumina Miseq platform (2 x 250) using the nextera xt kit for library preparation. So far I have removed the nextera primers with Trimmomatic and have assembled the genomes de-novo using SPAdes, IDBA and CLC, with SPAdes yielding the best assemblies based on Quast analysis. The assembled genomes consisted of ~100 contigs (100 bp cutoff). I also annotated the genomes using the Prokka pipeline.

What would be the next logical steps to take if I want to compare the genomes with the reference. Can this be done visually using the contigs, or should they first be re-ordered/align with closely related genomes. If so, what suitable software can be used?

Any help/guidance will be greatly appreciated. Thank you in advance.

[GenoMax]

What would be the next logical steps to take if I want to compare the genomes with the reference.

Try Mauve.

Answer 1

[Mensur Dlakic]

You have done well, assuming that proper methodology was followed.

I am not sure why you are expecting that genome sequencing will yield any differences, as culturing for several generations under different stress conditions usually is not enough to impart much change. If we assume that you mostly want to compare similar strains, you may want to look into pangenome analysis - both Google and PubMed searches will give you plenty of material. To get you started:

• http://pgaweb.vlcc.cn/

[JJ13]

It is an arctic soil isolate that has been cultured for close to a 1000 generations for each stress condition so the end goal would be to mine the genomes for any intra/inter specific changes, although I agree that I am not expecting any significant changes on DNA level. Will also do some transcriptomic work later one, but first just want to do a basic comparison of the genomes illustrating visually that they are either similar to the reference or that there are any/meaningful nucleotide substitutions/deletions etc. Thank you very much.