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Thread | Thread Starter | Forum | Replies | Last Post |
Miseq v2- Low cluster density, Low PF%, what should we do? | Joanna | Illumina/Solexa | 22 | 08-30-2017 11:30 AM |
MiSeq run with low Q30 and high cluster PF | haroldnunez | Illumina/Solexa | 12 | 01-06-2017 06:34 AM |
Newbie question: low cluster density on MiSeq (Chip-Seq pool) | HelenaSC | Sample Prep / Library Generation | 12 | 07-18-2013 07:28 AM |
MiSeq v2 cluster density 1/2? | pmiguel | Illumina/Solexa | 37 | 03-05-2013 03:33 AM |
Low cluster density of Truseq "high throughput" libraries - both DNA and RNA | SLeigh-Brown | Illumina/Solexa | 0 | 01-07-2013 03:19 AM |
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#1 |
Junior Member
Location: Perú Join Date: Oct 2017
Posts: 3
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Hello everyone.
Please, I need a help with my MiSeq genome sequencing run. I am sequencing M. tuberculosis genomes using Nextera XT and a 500 cycles v2 cartridge/flow cell. I always obtained aproximately 950-1050 K/mm2 and a passing filter more than 85%; However, in my last run I obtained 1063k/mm2 (i think it is ok), BUT JUST 50,3% %PF... sincerely, I dont have any idea what happened. I know with overclustering there will be a lower %PF, but in think is not my case. I s the first time i have this issue. Thank in advance for your answers. |
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#2 |
Member
Location: Europe Join Date: Oct 2016
Posts: 42
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Hello,
take a look at the cluster pictures. If you overcluster to much, the MiSeq cannot detect all clusters correctly and reports a much smaller number. fin swimmer |
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#3 |
Senior Member
Location: East Coast USA Join Date: Feb 2008
Posts: 6,699
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1000K/mm2 is outside limit per sepc for V2 chemistry. While it is tempting to push cluster density (trying to get additional data) the fall in data yield/quality is precipitous when you go over a certain density (like you discovered). You would want to carefully look at the data you have to make sure there are no artifacts.
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#4 |
Junior Member
Location: Denmark Join Date: Oct 2017
Posts: 6
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Have a look at your intensities of the first 25 cycles (e.g. in the sequence analysis viewer) (which might not only be a problem for amplicon sequencing). Below a quote from a Illumina white paper:
The %PF calculations involve the application of a chastity filter to each cluster. Chastity is defined as the ratio of the brightest base intensity divided by the sum of the brightest and second brightest base intensities. Clusters “pass filter” if no more than 1 base call has a chastity value below 0.6 in the first 25 cycles. |
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#5 |
Junior Member
Location: Perú Join Date: Oct 2017
Posts: 3
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Thanks for your answers ... I read all of it., I got surprised because at two previous runs the last year I got a cluster denstiy of 1200 and 1100k / mm2 aproximately (definetly an overclustering) but the passing filter was approx 80%. Then I recalculated my steps and I corrected that. But now, I think I did not get it too much (just 1069K/mm2), and the passing filter fell too much.
Now I am suspecting for the machine, MISeq. Because other people used to have the same, even worse, results with passing filter fell until 15%. Now I taked a look for my intensities at the first 25 cycles (like "provirus" recomendation) and I notice some extrange: first, definetly i got overclustering; second, the "T" base shows a marked overclustering (see picture), and thats the same result for all the run. could there be a particular reason for such behavior? For now, given the picture intensities Will it decrease my input DNA from 20 ul to 18 ul? Thanks for your answers and time. |
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#6 |
Junior Member
Location: Perú Join Date: Oct 2017
Posts: 3
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whats i hapennig... all my replies never come out
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#7 |
Senior Member
Location: East Coast USA Join Date: Feb 2008
Posts: 6,699
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Tags |
miseq, passing filter, problem |
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