We're planning to next-gen sequence flow-sorted microbes out of sputum samples. Flow sorting will be done using a fluorescent antibody. Our concern is that the staining involves a formalin-fixation step, and that this might crosslink the microbial DNA, making it useless for DNA sequencing. Does anyone have experience sequencing antibody-fixed/sorted microbial DNA? We'd like to know if there are any pitfalls to this approach before we invest the money. Thanks in advance.
Hi Christian, sorry this is not an answer. I'm also very interested in the feasibility of this method, but applied to fungi only. Are you selecting for bacteria, fungi or both?
Hi James - glad others have similar interests. We'd like to enrich for a specific sp of fungus in patients with fungus-induced pneumonia. Ideally we'd get rid of most human and bacterial DNA. We're hoping to get as many high-quality sequencing reads out of our run as we can.
Hi there,
if you are dealing with cell-walled microbes (bacteria, most fungi) it shouldn't be problematic to flow-sort them without prior fixation (although you could try to improve the staining by using 50% EtOH). A simple staining with DAPI, SybrGold or a Syto stain should do. Don't sort them into distilled water, or your cells might get lost.
Hi Christian, I've been poundering about something similar and hence don't have a clear answer. As you are interested in enriching for a specific fungus and if this would be possible, I was wondering if it would also be possible like to subdivide a large heterogenous bacerial population into different subpopulations using a flow sorter?
Hi again, another question to add to the mix. Is it possible to sort and isolate a single cell, in such a way that it is viable (i.e. could grow on a plate) or can be used for single-cell sequencing?
James, I can recommend the following paper:
Specific Sorting of Single Bacterial Cells with Microfabricated Fluorescence-Activated Cell Sorting and Tyramide Signal Amplification Fluorescence in Situ Hybridization, Anal. Chem., 2011, 83 (19), pp 7269–7275. So I would think it is possible.
Christian - Thanks for your response. I've been reading today and think you're absolutely correct. The protocols I've seen use a fixation step if you stain and cannot run your cells within an hour or so. However, if one could stain then do FACS immediately a fixation step shouldn't be necessary. This would allow you to get viable cells out of the sorter. Do you have any ideas of what to use instead of distilled water? Isotonic solution?
Koenraad and James - Thanks for your help. I found a similar paper: High-speed cell sorting of infectious trophic and cystic forms of Pneumocystis carinii, J Eukaryot Microbiol. They use FACS to isolate viable and infective Pneumocystis populations. Those cells should be healthy enough to be cultured (if Pneumocystis could be cultured, which it can't) or DNA Seq.
In our case PBS did work. Somehow distilled water worked like a black hole.
You might wanna have a look at the publikation list of Ramunas Stepanauskas (single cell sorting, and whole genome amplification). If you let them do the work it will cost you a bit though.
http://www.genomeweb.com/sequencing/focusing-high-throughput-single-cell-genomics-bigelow-looks-add-sequencing-its-s
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