How can I get or create a reference genome for Bacteria?

Question

I am a computer engineer and nowadays trying to grasp some concepts of Bioinformatics particularly, reference genomes and genomic variants. My aim is to find the effect of sequence features on variant types that occur in bacteria genomes. I chose Escherichia coli as the bacteria for my study since there seem to be more publication and data about it and its genomic sequences.

When I made a research about variants, I found out that we need to have a reference genome for the comparison with other alleles in order to find variants. And, I even read that we can use genomic sequence of one bacteria as the reference but it won't represent the cohort in an accurate manner. I got the genomic sequence data for several strains of Escherichia coli bacteria that been sequenced before. However, most of these sequences are not complete ones. I filtered out the complete DNA genomes. When I checked the reports on these genomes, I learned that these are complete genomes but not reference genomes. Only the ones which have RefSeq category: Reference Genome in their report are reference genomes for Escherichia coli.

Learning that most of the data I got for different strains of Escherichia coli were not reference genomes, I tried to seek to find reference genomes and I got this website of NCBI ref sequences for prokaryotes. This website shows only two reference sequences which match with what I have find in all sequencing data I got before. So, I want to ask two questions

First: How can I get a reference sequence for other strains of E. coli?

Second: If I cannot get a reference sequence for other strains, can I use complete genome data as the reference one? When I check the complete genome data, I cannot understand their order because there are other data for same strains that show loci of sequences but in the complete genome, I cannot see any order of the loci. Even, I cannot see data of some loci in the complete genome which blows my mind more.

I know it may not be appropriate to ask two questions in a single post, however, these two questions seem to support each other. I will appreciate any of your help.

Answer 1

How can I get a reference sequence for other strains of E. coli?

You can use any sequence you want as reference for a variation analysis. It just depends on the question you want to answer.

So to keep it simple, your Question is:

What are the variant region from a fresh sequenced E. coli K12 sample?

To know the differences from your known strain E. coli K12. A reason for this could be that you work with the strain K12 through out several years. During your studies you changed the conditions for the bacteria, like adding antibiotics or change the growth media. So some of the E. coli would adapt to the antibiotic and become resistant to it or they adapt better to the new media. This happens because some nucleotide changed. With each new experiment you used the best adapted colonies from the previouse one.

So you sequence your bacteria and compare it with the E. coli K12 strain. In this case this is your reference. If you use another strain, like SE15 or SE11, as reference, your result would not give you any answer for your question.

If your used data and publications gives you the information which strain were used, then use the given strain as reference.

Answer 2

in genomics, organisms have a single reference genome which is updated( or not) over a period of time. E.coli has different strains and this means there might be reference genomes for each of the strain. If there is no reference genome available the its likely that in constructing the sequence of those strains they might have used any of those two as their reference. This information you can find by reading the paper that published those strains. Therefore I will advice you pick any of those two available reference genome as your reference and work with and make sure you indicate which one you used in your report or document.