Junior and Flx compatibility

[ngseq]

Has anyone used Junior to sequence library made using GS FLX Titanium emPCR L kit? We tried doing so as our FLX sequencer is down but found only very few reads passed filter (even for control beads). So I am guessing there is some compatibility issue?

any thoughts please?

thanks!

[RCJK]

AFAIK the chemistries should be completely compatible. What type of library was it and what filters did the reads not pass?

[ngseq]

Thanks RCJK.

It is a shotgun ligated library.

here is what I got in the QualityFilterMetrics for the bad run (note control beads failed just as badly):

key CATG GACT
NumKeyPassed 261 248226
NumDotFailed 6 30740
NumMixedFailed 68 111699
NumTrimmedTooShortQuality 109 102992
NumTrimmedTooShortPrimer 0 437
TotalPassedFilter 78 2357

I re-ran emPCR using the Junior emPCR Titanium kit and got much improved result. Here is what the QualityFilterMetrics file looks:

key ATGC CATG GACT
NumKeyPassed 4054 2732 134569
NumDotFailed 1 4 2157
NumMixedFailed 59 26 10656
NumTrimmedTooShortQuality 27 265 18689
NumTrimmedTooShortPrimer 0 0 301
TotalPassedFilter 3867 2437 102746

Any clue what went wrong? Thanks!

[RCJK]

Hmm...I'm not too sure. For the bad run there also looks to be a high level of mixed reads that were rejected. This can be due to a few things: the library not being denatured prior to addition to the emPCR, too high a cpb leading to too high an enrichment, broken emulsions. Perhaps any or all of those occurred and maybe this also would affect the number filtered for short quality? That one I'm not very sure of but maybe someone else has some thoughts?

[pmiguel]

[...] too high a cpb leading to too high an enrichment, [...]

Is it well known that if your cpb is really high (like >10x) you can end up getting enrichment percentages that look acceptable?

In such cases, I think the numbers of positive beads overwhelm the system. Positive beads get lost because there are not enough enrichment beads to immobilize them all.

There are clues during enrichment that something is badly wrong. But if you ignore the odd behavior and look only at the final %enrichment you can be led astray.

I ask because we have run into this on both the SOLiD and the GS-FLX. But, best I can remember, I have never heard any warnings from Applied Biosystems or Roche about this possibility.

--
Phillip