I have a collection of bacterial isolates from which I want to obtain the plasmid sequences. I have two options:
1. Extract the plasmids and then sequence them
2. Subject the bacteria to whole genome sequencing and then extract the plasmid sequences.
Will the two give comparable results; if not, which is better?
If I go for option 2, I will appreciate the step by step procedure and the available free softwares to do this.
Thanks
kayode
Please consult the following paper:
Article Comparison of six commercial kits to extract bacterial chrom...
I think either approach would work, but if you only want the plasmid sequence it seems much easier to just isolate the plasmid and sequence it directly. That will simplify the subsequent bioinformatics steps.
It is simply to isolate plasmid and send it to sequencing, also the plasmid can be transformed to competent cell (e.g. DH 5alpha E. coli) and give details about your plasmid and inserted sequence
I agree with Mathew A Beale , extract the plasmids and then sequence them in a long read platform
I don't know if this answer will be in time for your research, but it may help others who encountered the same problem.
The answer is depends on what you know/want to know about the plasmids.
In my case, I started with a brand new isolate that I don't know whether it carries a plasmid, number, and size of the plasmid(s), if present.
- I started with using a plasmid extraction kit, measuring OD260 of the extract, and run the gel to see if there is any band(s) of DNA that looking like plasmids, and estimated the smallest plasmid size (I used supercoiled DNA size marker).
- Then, I used the Wizard genomic DNA extraction kit (it will also extract plasmid DNA at a good ratio to the chromosomal DNA, see the paper Kandasamy Ulaganathan recommend.
- Then conducting the whole genome sequencing using both long-read Nanopore and short-read illumina platform. Then do the hybrid assembly (look in https://galaxyproject.org ). You should get a good and accurate sequence of both chromosome and plasmids of your unknown isolate. (only the short-read is not enough to identify which contig is plasmids or fragments of the chromosome. You can do only Nanopore, if you are not serious about accuracy).
- This approach will give you all the information you need, especially if you know nothing about the plasmids and also get information on the bacterial genome itself as a bonus
- **Be careful** I recommend Nanopore since PacBio will only give the sequence of large plasmids (in my case, it can't detect plasmids smaller than 6 kb). This is where the gel electrophoresis come into play, so you can check the sequencing result whether it covers the smallest plasmids the strain carries or not. Look into the PacBio Protocol, it aims to select only large fragment, at least >10 kb up.
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