Hi,
My lab is getting prepped up to do genome sequencing of several S. pneumoniae and N. meningitidis isolates. We got far better growth for S. pneumoniae with 5% Sheep blood serum in Todd-Hewitt broth with 0.5% Yeast extract. However, same preparation did not work for N. meningitidis.
We are using Qiagen Blood Mini Kit for extraction.
My concern is - does the serum anyhow affect the quality of genome sequencing? More to that, I would really appreciate your suggestions for growing N. meningitidis in liquid broth.
Thanks in advance. :)
Hi Arif,
Most DNA in the serum will be lost when you centrifuge the cells at the start of your DNA isolation. If you are particularly worried about this you could introduce an extra wash in PBS or treatment of the cells with DNase before lysis.
I've never heard of commercial horse serum being DNA-free and though it won't have DNA containing cells, I wouldn't assume it to be DNA-free.
Thanks, Dr. Beale.
Actually, that was the part I am a bit worried about (the serum may contain sheep DNA). But, you are also right that aligning sequence with reference genome would solve the problem. Nonetheless, we are checking all the possibilities. We also have commercial Horse Serum (Gibco, Life Technologies). This should be DNA-RNA free, right ?
Hi Arif,
Most DNA in the serum will be lost when you centrifuge the cells at the start of your DNA isolation. If you are particularly worried about this you could introduce an extra wash in PBS or treatment of the cells with DNase before lysis.
I've never heard of commercial horse serum being DNA-free and though it won't have DNA containing cells, I wouldn't assume it to be DNA-free.
Thanks Dr. Brouwer.
So, I will proceed with Sheep serum, which will be more cost-effective (we have our own sheep).
Thanks a lot for your help here.
Hello Mr. Arif,
As per the suggestions given by the two people above, I agree with them and like to stress my experience on NGS from P aeruginosa from culture plate.
Since you going to culture them in a Blood agar plate, You need to get higher quantity of DNA (Depends on platform and kit it varies too) You take enough colonies from the plate and wash it with buffers by centrifugation.
90% Sheep blood contents will go off, but still if the sheep DNA present in your bacterial DNA, this will overcome your NGS data.
In this scenario, You must produce at least 500MB data, to get good quality results for exclusion of sheep genome while analysing QC for NGS.
Alignment with reference genome will fetch you good results.
All the best.
Regards
Murugan N
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology