I have just sequenced two different dilutions of the mock community HM-782D. For one of these mocks I followed the manufacturers instructions and didn't dilute my sample. For the other I followed the instructions except I used a 1:1000 dilution. My undiluted mock came out OK but my diluted mock came out full of contamination.
As I am looking at lung bacterial communities I am a bit nervous about contamination due to the low bacterial biomass in my samples. I ran negative controls for all of the reagents I used in my PCR. However, the sequences I am seeing in my mock dilution do not appear to correlate with those from my negative controls.
I did think that I may have got a lung sample mixed up with the mock as there is some crossover between the types of bacteria I am seeing in the mock and those I sometimes see in lungs. However, I have checked and no lung samples were processed on the same day as the 1:1000 dilution so this seems unlikely. Does anyone have any other potential explanations? Might it be contamination from the lab that made the mock community?
Did you use the entire sample to dilute, or do you have some remaining that you can test undiluted (assuming undiluted and diluted already used were from separate vials) to rule out contamination from the original lab?
They were made from the same vial so that isn't possible unfortunately.
Though it is very silly to ask, have you checked your reagents used in dilution (water or buffer) ? If no, then better to cross check microbial contamination in reagents used in dilution.
Usually, the bacterial contamination is a sterilization problem, if your undiluted sample had no contamination, I think there is some sterilization chain is broken.
Check your procedure, step by step. If you are sure that you have not done anything wrong, it might be because of the reagents used. Did you sterilize the diluent as well as the pipettors/tips? Has the area been cleaned/disinfected before you do the experiment? Where did you store your sample? Have you done this on the same day of the sample with no dilution? These are the things you might want to check.
1. The only thing different between the 'non-diluted' and 'diluted' mock samples are the 'solution' used for dilution. You have not mentioned whether you had checked this solution.
2. If this lab has been intensively used as a place for the study of 'bacterial community' from lung for a while, the tools such as pipets might have been contaminated with those bacteria (during pipetting). So, if you used these pipets to do experiments, such as transferring samples, dilution,...etc., even "no lung samples were processed on the same day", the early contaminated bacteria might have cross-contaminated into your new preps. The powerful PCR can pick up even trace amount of contaminated bacteria and their DNA.
They were diluted using the nuclease free water I used for my negative controls. After checking back though, the 1:1000 dilution was prepared after all of the other samples so contamination from other samples is possible.
Did you sterilise the nuclease-free water by a validated method prior to use? This is the most likely source of your contamination. Otherwise I agree with the comments of all colleagues above.
http://www.biomedcentral.com/1741-7007/12/87
Did u sequence by any chance on a miseq/hiseq, how was the data analysed ?
I always UV my nuclease free water for 30 minutes before I use it for qPCR and PCR and run negative controls for each PCR. In the past I have found that the water can be a source of contamination which shows up after 30 cycles of PCR.
You may want to compare the total read numbers for your diluted mock, undiluted mock, and negative controls. There is a good chance that your diluted mock has low read number (similar to your neg control)? I thought 10 fold dilution is fine, but 1000 fold dilution?
In answer to people's comments, I sterilise all of my reagents (apart from those that would be damaged) with very strong UV for 30 minutes. My DNA is sequenced in a Miseq and is processed through the mothur Miseq pipeline.
I agree that the amount of template I would be adding would be very small for the 1:1000 dilution and that it might be close to my negative control. However, in that case then I would have expected the same types of contamination in my negative control as in my 1:100 mock dilution.
Keep in mind that everything is a source of potential contamination. Not just the PCR reagents but extraction kits, tubes, etc. And the contamination profiles can change from lot to lot.
http://www.biomedcentral.com/1741-7007/12/87
So the explanation could be as simple as using a different bag of tubes to dilute the control. However, I think your comment about cross talk between your lung samples and the mock may be closer to the mark. We recently looked at indexing as a source of potential contamination - this was not a rigorously controlled study ;-) The run was a MiSeq V3 with about 25 bacterial genomes. We filled up the sample sheet to a full 96 with imaginary samples (dual indexed). Every one of those imaginary samples received around 200-300 filter pass paired reads. These were confirmed to have originated from the real samples on the run. Obviously this raises more issues with interpreting 16S data particularly for low level taxa.
I routinely use DNase away to decontaminate the bench surface before and after work with respiratory samples, or pipetting reagents for PCR.
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