I have been trying to perform phage purification (CsCl density centrifugation), DNA extraction (Norgen phage DNA isolation kit) and phage sequencing (MiSeq PE-150) for therapeutic phages targeting Pseudomonas aeruginosa.
The phages were first isolated by lab strain and later trained to infect patient's Pseudomonas aeruginosa bacterial isolate.
But many of the times, I seem to have only prophage sequences from patient bacterial isolate, which are not what we isolated at first.
May I check if this occurs commonly?
Did I do something wrong during phage propagation? Or is there anything to take note during sequence analysis pipeline?
Robert Adolf Brinzer Thank you for your reply. I think I might not have phrased my question clearly at first. Have amended it.
The phages were isolated by lab strains at first, and then trained to infect patient bacterial isolate. However, our sequences came back to be all prophage sequences from patient bacterial isolate. So it seems that all my original phages have been lost. I am wondering if this is a common occurrence, or is there something that I am doing wrongly for phage propagation or sequence analysis pipeline? Thank you again.
Shuhua, I am still confuse by what your results actually are. Are you saying that you began with a pure phage stock whose genome sequence you know, but upon infection of a strain you no longer recover that phage?
Please describe in more detail your experiment.
Michael J. Benedik
I began with a pure phage stock isolated using a lab strain. The genome sequence is not known yet.
I used this phage to train against patient bacterial isolate to make it propagate better in patient isolate. After a few rounds of training, I attempted to purify the phages I got (propagated using patient isolate) to send for sequencing.
But the sequences came back to be just prophages found on clinical isolate. No signs of other phages.
I am wondering prophage induction happened here? Are the occurrences of prophage induction are high? And if there are ways to avoid them?
Prophage sequences are seen for many of my other Pseudomonas phage sequencing samples as well.
It is certainly possible that you got a phage released by prophage induction. However normally that would not form plaques on a strain which is lysogenic for that phage, which is the assumption you are making. But it is possible the phage picked up a mutation whereby it won't be repressed by the lysogen.
Did you do a control experiment without adding your isolated phage first to see if you recover the prophage plaques?
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