jmeskas at bccrc.ca
Thu Jun 26 01:09:43 CEST 2014
Hello Kipper and Pratip,
Thank you for your explanations. After looking at my data more closely, I found that about half of the cases where flowClean was removing only the first compartment were consistent with the shape of the data. The other half of these files seemed to just remove the first compartment randomly. I have created an R source code file you can use to replicate this result. I have put it into a .tar.gz file and will transfer it to you from my google drive in a follow up email. Please do not redistribute the data. Inside the .tar.gz file there is a folder called Figures that can be regenerated using the code. The figures in Figures/Clean show the output of flowClean, while Figures/CleanTest show plots of Marker Vs Time that I created using plotDens from flowDensity. (I am using these Marker vs Time plots to judge if a certain section of the data should be removed or not.)
Files "SPLN_L000030297_P3_090.fcs" and "SPLN_L000031107_P3_141.fcs" show when flowClean has removed the first compartment when I believe it should not of been. The other 5 FCS files show cases where flowClean seems to also give poor results (The other non-first-compartment-removed files all looked good). In my opinion, flowClean should be removing, from the following files, the following sections:
SPLN_L000018651_Size_113 - 0-5% marks
SPLN_L000018653_Size_115 - 0-5% and 75-80% marks
SPLN_L000018656_Size_118 - 0-5% marks
SPLN_L000019881_Size_148 - 0-5% and 20-25% marks
SPLN_L000028450_P3_054 - 0-5%, 12-17% and 55-60% marks
SPLN_L000030297_P3_090 - Nothing
SPLN_L000031107_P3_141 - Nothing
For SPLN_L000018653_Size_115, SPLN_L000018656_Size_118 and SPLN_L000028450_P3_054 there seems to be certain locations where only one marker is having a problem and it is not removed. Is it the case that flowClean does not consider 1 marker problems to be substantial enough to remove?
Any insight you might have on any of these problems would be greatly appreciated. Thank you very much,
P.S. I have made the code, hopefully, easy enough to use so all you have to do is change the working directory to the folder that the files have been extracted to. Let me know if there are any problems with the code.
From: Pratip K. Chattopadhyay [pchattop at mail.nih.gov]
Sent: June 25, 2014 7:07 AM
To: Kipper Fletez-Brant
Cc: Justin Meskas; Ryan Brinkman; bioconductor at r-project.org
Subject: Re: flowClean
There are probably a couple of factors at work here...
The HTS is more likely to exhibit anomalies early in collection for various reasons... The pressure in the system may still be building up, the cells are settled in the bottom of the well and so more events go through at once, clogs/debris from previous wells/runs may dislodge. In principle, the system is engineered to avoid these issues, but in practice, I often (but not always) see anomalies at the beginning of the collection. Interestingly, on days/runs where there aren't many bad regions flagged, the early regions also look good. This inspires confidence that the algorithm is detecting true problems and doesn't have some systematic problem.
The second factor - relevant to the case where you felt the first events weren't too bad - is guilt by association. Kipper has built in a little buffer to take out some bins that neighbor trouble spots, just to keep things as clean as possible.
[cid:part1.07090309.09090509 at mail.nih.gov]
Kipper Fletez-Brant<mailto:cafletezbrant at gmail.com>
June 25, 2014 8:56 AM
We (Pratip and I) think it may likely be your data - we have observed that the early time points of collection in a flow run tend to have the most errors. Pratip can speak a little more to the technical causes of this. We appreciate your comments and look forward to the results of your tests.
On second thought, I think it is my data. I just checked a few files and they seem to be consistent with only removing the first compartment. I will run some tests tomorrow to validate this. Sorry for the emails.
From: Justin Meskas
Sent: June 24, 2014 4:31 PM
To: Kipper Fletez-Brant
Cc: Ryan Brinkman; bioconductor at r-project.org<mailto:bioconductor at r-project.org>
Subject: RE: flowClean
Sorry to keep emailing you, but I had another question about flowClean. I have been noticing that the clean function seems to label the first compartment for removal every time. This seems odd to me. I attached two figures. The figure called "A..." looks like most other figures, where the first compartment is labelled for removal. And the other figure, called "B...", is my unique case where, I believe anyway, the first compartment should be removed, but not the second. Are all these files somehow accidentally removing the first compartment? Or do you think it is the case that all these files have bad data at the beginning?
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