Originally posted by mcnelson.phd
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Originally posted by pmiguel View PostWhy are phasing/pre-phasing calculations affected by low diversity? I can't think of a mechanism. Unless it is just follow-on issues from bad matrix calculations. -- Phillip
I'm sure someone with more knowledge than myself can give a better explanation of that.
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Originally posted by pmiguel View PostWhy are phasing/pre-phasing calculations affected by low diversity? I can't think of a mechanism. Unless it is just follow-on issues from bad matrix calculations.
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Phillip
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Instructions we just received for using a hard coded matrix and phasing appear to be different than the ones listed here. We are going to try them out today.
BTW: What version of MCS are you running?
Originally posted by Vinz View PostInstructions for hardcoded phasing/matrix with upgraded MiSeqs:
Choose a good run (phiX).
Run folder\Data\Intensities\Basecalls\Matrix\s_1_1_matrix.txt
Run folder\Data\Intensities\Basecalls\Phasing\s_1_1_phasing.txt
Copy these 2 files and rename them as hardcodedmatrix.txt and hardcodedphasing.txt respectively.
Place them in C:\Illumina\RTA
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I'm wondering about hardcoded phasing/matrix files...
In the blog post here (http://pathogenomics.bham.ac.uk/blog...llumina-miseq/), it is indicated that after 10/31, you don't monkey with the .xml file, just save your "nice" phasing and matrix files as phasing.txt and matrix.txt into the RTA directory. I finally ran some 16S runs in January, and I'm not convinced the instrument fell back to the desired values as I had exceptionally high phasing/prephasing. The first run gave the following phasing/prephasing:
300cycles, 50% PhiX, read1: 0.104/0.533, read2: 0.298/0.610
There was a massive intensity spike during read2 (and the qscores not so good), so our FAS sent us a replacement kit. I reran it, but decided to try the "custom read" primer wells, and so I don't know the phasing/prephasing values are trustworthy. But despite losing my PhiX-based metrics by doing this, I assume the complexity was still observed, so they can't be too far off. Also, this was run the day of or the day after the most recent software upgrade, and I realized after the fact the upgrade had removed my phasing/matrix files (as well as my preference to generate an index read fastq):
300 cycles, 25% PhiX, read1: 0.581/0.234, read2: 1.146/0.000
So that run also looked terrible and our FAS sent us another kit to try. The data for read1 look much better this time with most reads at or above q30 at 90% read length, but the phasing/prephasing still look bad. Also, quality took a serious dump on read2:
300 cycles, 25% PhiX, read1: 0.094/0.573, read2: 0.380/1.160
Our genomic runs all look great, so I know this has everything to do with low complexity. Can anyone confirm that with the latest software upgrade we just drop new phasing.txt and matrix.txt into the RTA directory to correct this problem? Any other manipulations of the software that people have found to help?
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I was explained that the difference between modifying the MiSeqConfiguration.xml file and adding the matrix/phasing files is the following:
1. MiSeqConfiguration.xml set hardcoded values will always be used (Nicks post is still up to date for the current release)
2. matrix/phasing txt files will be used in case the software assumes that something with the calculation of matrix/phasing is wrong. Meaning: In extreme cases both methods work the same. However, with version 2 you may run in the situation, that you have used amplicon but RTA has decided to use the calculated phasing/matrix values. That may not give you good results. You know after the run: If the displayed phasing values are those that you have set, it was hardcoded. Otherwise it was not hardcoded.
We prefer version 1 for amplicon sequencing. That seems to work pretty well.
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I do not think a restart of MCS is required to apply a new MiSeqConfiguration.xml file. My understanding is that it will look for the xml at the beginning of the run.
However, if you want to do a run without hardcoding, you would have to change it back... That's definitely true.
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Thanks for the advice.
Just to confirm, you can still name the new files "hardcodedphasing.txt" as long as the xml is telling the software what to look for? Or should I plan to just rename files and restart MCS prior to each run if I am switching from high to low complexity templates?
Also, since our first run had phasing and matrix files in place but the phasing/prephasing didn't match the PhiX values, and PhiX was a bit high (50%), it seems to me that if you plan to use hardcoded values then lower PhiX is warranted.
Finally, if I want to keep the image files to toy with different settings (<CopyImages>true</CopyImages>), does anyone have an idea how much space this will require from a single 300 cycle run? Our Miseq only came with a 500GB drive and I don't want it choking half-way through a run. I did network it (gigabit connection) to our local RAID device which has much more space, so I could place the output there, I suppose, but sometimes the network can screw you as well.
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Originally posted by Vinz View PostI do not think a restart of MCS is required to apply a new MiSeqConfiguration.xml file. My understanding is that it will look for the xml at the beginning of the run.
However, if you want to do a run without hardcoding, you would have to change it back... That's definitely true.
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If you want to follow option 2, then you do not have to change the xml. Then lower PhiX may give you better result as high PhiX might prevent hardcoded phasing and still have bad values. If you want to follow option 1, then change the xml according to Nicks post. If you save a xml version with and without hardcoding, you may switch between those by renaming the files.
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Originally posted by AKrohn View PostThanks for the advice.
Just to confirm, you can still name the new files "hardcodedphasing.txt" as long as the xml is telling the software what to look for? Or should I plan to just rename files and restart MCS prior to each run if I am switching from high to low complexity templates?
Originally posted by AKrohn View PostFinally, if I want to keep the image files to toy with different settings (<CopyImages>true</CopyImages>), does anyone have an idea how much space this will require from a single 300 cycle run? Our Miseq only came with a 500GB drive and I don't want it choking half-way through a run. I did network it (gigabit connection) to our local RAID device which has much more space, so I could place the output there, I suppose, but sometimes the network can screw you as well.Last edited by GenoMax; 02-08-2013, 08:39 AM.
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Anybody using 500 cycle kits for amplicons sequencing? Really need to make that work to completely supplant GS-FLX amplicon reads.
Also, it was my understanding that the new MCS version 2.1.1.13 had some improvements for handling low diversity data. Anyone seen evidence of this?
--
Phillip
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