recently, i sequencing the plasmid in E. coli with the next generation sequencing. But, unfortunately, the contig is no longer than 5 kb. The depth of the sequencing is 1 GB for a sample. what is the reason of it?
How large do you expect the plasmid to be? There are several plasmids known to be present in E. coli of around 5 kb. So if you are just looking at which naturally occuring plasmid is present, the simplest explanation would be that your plasmid is around 5 kb long.
Are you sure the plasmid was pure and you did not have any low level chromosomal DNA? What was the average read quality and read length and on which platform did you sequence?
It is impossible that the plasmid is around 5 kb. And the contamination of chromosomal DNA could not be avoid after many attempt.
Hello Yuqing,
What platform of NGS are u using.
You can check your plasmid size on a gel and if it is around 5 kb then your problem is solved. Genomic DNA will cause u big time problem if it is their. Try to load your plasmid in a gel and elute it from the gel before going to NGS. It will give you good plasmid.
Is 5 kb your largest contig?
What is your total number of contig?
Dear Nithin,
The platform we are using is HiSeq XTen. And the plasmid is larger than 5 kb. Our largest contig is 5 kb, which confused me for a long time. And the number of contig is 150.
that is quite a lot of contigs for a plasmid (even fo a whole genome of E.coli I would like to have less). What is your overall read length, your read quality. Did you use paired ends? Was the fragment size ok after fragmentation, not too many small fragments? Which assembly algorithm did you use?
These are all elements you need to look at to pinpoint the problem, but if I have to guess based on the information you already provided, I would guess you have a quality issue with your DNA that needs to be cleared up prior to sequencing. Are you sure there is only one plasmid in the sample? Or can there be multiple, similar plasmids?
There are at least three plasmid in just one strain according to the analysis of PlasmidFinder. The fragment size ranged from 300 bp to about 1000 bp.
if the three plasmids have a relatively high similarity, you will need to seperate them on gel first. You will never get a good assemlbl with different, but closely related plasmids.Alternatively, as you have an idea of which plasmids are present, you can try mapping your reads to the reference sequences of these plasmids.
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