Plasmid in gDNA - Will it influence my sequencing quality?

[illnoobina]

Hello everyone,

like my name says I'm a noob in sequencing and want to sequence 5 presumably E. Coli genomes. My whole setup is the following:

- gDNA extraction: Quiagen DNeasy Blood & Tissue
- Sequencing platform: Illumina MiSeq
- Library preparation kit: Illumina TruSeq
- Shearing: Covaris S2
- Quality Control: Bioanalyzer, Qubit, PicoGreen ...

I extracted my DNA and run it on a gel and found that 4 samples had an additional 2 bands running at 1000 and 1300bp. I assume that those bacteria contain a plasmid and the two bands are the plasmid in normal and supercoiled form.

My fear is that the sheared plasmid DNA might be the dominant species in my sequencing run and I end up with a poor read covarge on my genome.

Is this a problem and If yes is there any way I can fix it?

I searched the forum for a similar thread but didn't find one. So if there is an existing one just tell me and close this thread!

Thank you

[rnaeye]

Hi!,
Did you grow your E.coli in the presence of an antibiotic and select for plasmid containing cells? How do you know what you are seeing is plasmid? Can it be partially digested RNA? If it's plasmid DNA, yes you will sequence it. If it's as much as your gDNA, you should expect that half of the reads would be plasmid reads?

You can also gel purify your genomic DNA and make a library. You can digest agarose by β-Agarase. I have not tried it myself buy one of my colleague do it all the time for genomic DNA extraction from agarose. NEB has a library prep that allows you to make libraries as little as 1-5 ng input DNA. It's called "NEBNext Ultra DNA Library Prep Kit for Illumina". By the way, I believe Qubit dye cannot bind supercoiled DNA and you need to linearize it. Hope this helps.

[Brian Bushnell]

I extracted my DNA and run it on a gel and found that 4 samples had an additional 2 bands running at 1000 and 1300bp. I assume that those bacteria contain a plasmid and the two bands are the plasmid in normal and supercoiled form.

Have you isolated those bands and sequenced them, to determine what they are?

[illnoobina]

Thank you for your answers!
I have news: I repreped my gDNA since covaris shearing didn't work as expected. In the new DNA prep I did a RNaseA treatment and now both bands are gone. I assume that the bands weren't plasmid bands at all but were 23s & 16s rRNAs. My band patterns are very similar to those in A (not my image!):

https://norgenbiotek.com/product_images/water_rnadna_purification_kit__045_m-26400-figure1.jpg

I would love to upload my gel pictures but my program is very strict about plagiarism and I assume they will start picture comparison next ...

Hi!,
Did you grow your E.coli in the presence of an antibiotic and select for plasmid containing cells? How do you know what you are seeing is plasmid? Can it be partially digested RNA? If it's plasmid DNA, yes you will sequence it. If it's as much as your gDNA, you should expect that half of the reads would be plasmid reads?

You can also gel purify your genomic DNA and make a library. You can digest agarose by β-Agarase. I have not tried it myself buy one of my colleague do it all the time for genomic DNA extraction from agarose. NEB has a library prep that allows you to make libraries as little as 1-5 ng input DNA. It's called "NEBNext Ultra DNA Library Prep Kit for Illumina". By the way, I believe Qubit dye cannot bind supercoiled DNA and you need to linearize it. Hope this helps.

Hi rnaeye!
Yes one of the strains grew on AB but didn't show the two bands. I just assumed that it was plasmids displaying the bands. I think you are right about your RNA assumption since I didn't RNaseA treat my samples. I kind of assumed that I would work dirty enough to get rid of them .
And I Qubit my sheared DNA and PicoGreen my gDNA.

Funny thing is that it is possible to MiniPrep plasmids from these strains!

Have you isolated those bands and sequenced them, to determine what they are?

Hi Brian!
No I haven't done that yet but the plasmid DNA is preped now.

So if the bands are RNA my genomic DNA should be fine I guess. Now I have to rephrase my question: Is it possible to mix in my plasmids in the sequencing run? Should I give them individual indices or rather mix them to the gDNA of the strain of origin? I think giving them individual indices will be the better solution for the subsequent genome assembly?

Thanks again!
Illnoobina

[rnaeye]

If I had gDNA and plasmid DNA preps separately, I would barcode them and sequence them on the same flow cell instead of mixing them without barcode.

[illnoobina]

If I had gDNA and plasmid DNA preps separately, I would barcode them and sequence them on the same flow cell instead of mixing them without barcode.

That's what I meant by indivdual indices. So I build a library from each plasmid and gDNA and pool them for sequencing.

How would you pool the libraries? Let's assume the E. Coli genome has a size of roughly 5Mb and my plasmid has an estimated 5kb. So I would concentrate my gDNA 1000x in the library pool and the plasmids 1x?

[rnaeye]

Yes, you are right. If your goal is to get the same amount of coverage for each genome, you should put 1000x less plasmid DNA since it's smaller.