Dear researchers,
Hi,
I have sent a DNA sample of phage for Next Generation DNA sequencing (NGS) with HiSeq for a sequence with almost 40kbp size. The company has held the process to make sure if I’m ok with sequencing of smear containing DNA sample. I want to know, should I ask for continuing or sent them a new sample?
(QC image has attached, 1ul of sample DNA containing 130.195ng/ul, 0.5ul of sample has loaded onto 1% gel electrophoresis in 30min at 100V).
Sincerely
Alireza Mohebbi
From your QC image, i think quality of your DNA should be OK for NGS. The DNA is not smeared enough to affect sequencing.
the sharp band can be cut and purified from the gel, I think you can request the sequencing company to do that before sequencing
I would suggest that you should ensure purity before committing to NGS, the results may by poor otherwise. Do not take a chance.
From your QC image, i think quality of your DNA should be OK for NGS. The DNA is not smeared enough to affect sequencing.
Dear Guoqian,
Actually, I thought that way, but we are going to perform an other purification with viral nt acid purification kit. If the same result obtains, I ask for continuing the process with the same sample.
Running your DNA through a cleanup column is likely to do the trick. It does not appear to be of bad quality and you have a lot of it. The smear could be from bacterial DNA contamination. Given you state that you are doing HISEQ , then you should bnot have an issue with getting enough phage sequence back to assemble your genome
Hi
Your DNA is fine for NGS. Based on concentration provided, it seem that it might be contaminated, unless you are sure that virus was purified. You can verify it by using host specific primers.
After another purification perfomed by vral DNA extraction kit, the smear has resolved. And I think as you mentioned, bacterial contaminations would be the cause of high DNA concentrations and smears.
Bests
Though your sample has very minute smear, which may be a contamination of RNA or protein during your DNA extraction, can you specify the method of DNA extraction you have used.
Accordingly we can proceed.
Anyway the concentration of your Specimen DNA is more than enough to proceed for NGS step, since there will be a purification step during Library preparation is carried out. So nothing will happen if minute other traces present in it.
All the best in advance.
Dear Dr.Murugan
At first time, after phage propagation we have used ultracentrifugation and dialysis bag. In secong try, which led to a more clear DNA band. Invitrogen viral DNA extraction kit was used.
Best Regards
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