Antibiotics in lysis/wash buffers
H, In my recent ATAC-seq experiments, I am seeing some bacterial contamination, specifically pseudomonas, these are frozen postmortem human brains, so all dead cells. I am wondering if it would be a good idea to add antibioitcs in the wash buffer? If someone has experience with that, please let me know. Thanks!
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Hi, this is a common problem in NGS, which leads to bacterial reads come through in a NGS library. Hence, I wanted to see if someone had opinions on that.
I'd probably ask on SeqAnswers, as this lies right on the line where wet-bench biology meets bioinformatics. However, given that this involves details on wet bench procedure, it is definitely not something a bioinformatics specialist can be expected to handle.
I'd say the answer is no because antibiotics would only kill bacteria after a notable incubation time (hours) so 1) the DNA would still be in the mix, maybe require extensive washing, and 2) you cannot leave thawed tissue in any kind of culture for that long. After thawing it must be processed right away. Chromatin quality is probably abysmal anyway after a thaw cycle. Try to see whether this is really contamination or just BLAST artifacts (that is probably how you found it right, by BLASTing reads?) due to crap-quality libraries which probably come from abysmal chromatin quality of these thawed/death cells. As for the question being off-topic, well in part, towards the wetlab side, but if you want to discuss the strategy to detect the bacterial contamination that would I guess make in on-topic again, maybe change title and question a bit then.
Thanks ATpoint, I would actually really like to discuss 1) how to reduce bacterial contamination? 2)I have many reads that simply do not map to human genome, and upon using Kraken, they seem to match pseudomonas specifically, so I am not sure what you mean by BLAST artifacts?
Ah ok I see. People often blast like three unmapped reads and get spurious BLAST hits to random bacteria, and then say they have contamination, but you seem to have done it the proper way. I do not think that you can reduce contamination if you indeed have them in the sample. Probably chromatin quality is very bad due to the thawing so maybe this favours reads coming from minor contamination (thinking aloud). There were some papers starting from frozen tissue using the OmniATAC recently if memory serves, maybe you want to check them out. In the worst case you have to sequence deep/long enough to get enough on-target reads and simply discard the contaminant reads given that the on-target reads (so the ATAC-seq data) are actually fine and usable.