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Old 10-21-2014, 07:46 AM   #1
hoytpr
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Question Problems since long-read "upgrade". Just us?

So there may not be many of us left, but those who are still using 454-Junior machines... have you had as many problems with the upgrade to long reads as we have had? Before the upgrade, we never had a failure, now we can barely get a "+" run to pass specs. Sometimes the library looks whacky (technical term for a few different results). Sometimes the library looks okay but the run has very few reads that pass the filters.

The vast majority of the problem shows up as very high Trimmed too short, and this is followed by high Mix+Dots/Total Key pass. We've been getting advice from Roche, but mostly they are blaming the emPCR conditions. We've varied the conditions and even gone back to the old Titanium chemistry and run settings without much success. We don't have any FLX machine (or experience) so we aren't mixing those protocols that I know of. I tried a few configuration hacks for the filters, but nothing worked.

I'd like to hear of a couple success stories so we can emulate what you have done...
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Old 10-28-2014, 05:50 AM   #2
Anthony.287
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Hello!
I can't speak much about the Jr, but with the upgraded FLX I saw many odd results when mixing Titanium chemistry with +. For example, following the titanium emPCR instructions and sequencing using + yielded very poor results, while the same library with + emPCR and + sequencing, or titanium emPCR and titanium sequencing worked just fine.
Hope this helps!
Anthony
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Old 10-28-2014, 07:51 AM   #3
hoytpr
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Thanks Anthony,
We also tried to mix those chemistries without success, but I am much more concerned about potential software glitches, or that we have somehow just "missed" something. Dr Hong in my group is the library preparation specialist and he had been flawless over almost three years until the upgrade. I'm curious that no 454-Jr people are around to tell me the upgrade worked.
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Old 10-30-2014, 10:13 AM   #4
martin2
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Hi,
I spent 3 years analyzing public 454 data and found some artefacts and have several ideas how these emerge (seems multiple mechanisms are involved). Although I haven't published the details if I am to guess I would also say emPCR was the issue (or ... around it). ;-)
Would you disable in processing pipeline XML config files the length trimming I could look into the sequences you have. Ideally, provide me with 2 datasets fromthe sane PTP plate: one with "normal" processing and the other with those supposedly "short" reads obtained via loose processing. 10k reads are enough. Although I offer this as a paid service (including cleanup/rescue of the data), I could first check what you have got for free and provided I will succeed we can than negotiate what you can get back.
Martin
http://www.bioinformatics.cz
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Old 10-30-2014, 11:33 AM   #5
hoytpr
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Martin,
Thank you for the offer. I visited your website and it is very nice. Perhaps we can negotiate if no other people are able to help, and Roche cannot help either.

We will remember you and your services in the future.
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Old 02-02-2015, 12:59 PM   #6
mps4208
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Default Success on XL Junior!

I know this reply is way after the fact, hopefully you have had success by now. I write mostly because we were having the same issues and this seems to be the latest thread. We got the XL on the Junior to finally work with gDNA, haven't tried amplicons yet! It also took us about 5 tries, even though our regular length has been working for years.

Here is the multiple things that it took 1) Having the instrument serviced twice (valve manifold) 2) several lots of oil replaced 3) emPCR extension lengthened to 11min 30sec.
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Old 02-02-2015, 01:40 PM   #7
hoytpr
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Quote:
Originally Posted by mps4208 View Post
I know this reply is way after the fact, hopefully you have had success by now. I write mostly because we were having the same issues and this seems to be the latest thread. We got the XL on the Junior to finally work with gDNA, haven't tried amplicons yet! It also took us about 5 tries, even though our regular length has been working for years.

Here is the multiple things that it took 1) Having the instrument serviced twice (valve manifold) 2) several lots of oil replaced 3) emPCR extension lengthened to 11min 30sec.
That is interesting. Maybe we can request some new oil... that was a problem in the past if I recall. May I ask how you finally decided on 11 minutes and 30 seconds for emPCR extension?

Five tries is expensive. We had to stop after two bad runs because it ruined our Core Facility budget.
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Old 02-03-2015, 05:22 AM   #8
mps4208
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Default 11min 30sec

(It was expensive in time only as we got reagents replaced)
Roche tech support suggested since our rapid libraries were long 1,700bp (Agilent) that we might need 1-2 extra minutes during extension to ensure attachment to the beads and extension because of length. I choose average and added 1.5minutes to the emPCR XL cycling conditions.
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Old 06-22-2015, 03:13 AM   #9
GSviral
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Hey guys,

Recently I have performed 3 runs on the GS Junior using the long read kits after we got our machine upgraded.

I have ran into exactly the same problems as described here. Very low amounts of reads are passing the filtering process (only around 8%) and the reads that do pass are short reads - around 120 - 150 bp average.

I have also been in contact with Roche regarding this problem. I have sent off my library Agilent traces etc. to a Roche representative before I continue on to emPCR and each time the traces were pretty much perfect in size distribution and with no small fragments.

mps4208 - I tried your modified emPCR extension time and this still didn't seem to do the trick.

Have any of you guys got a definitive answer from Roche yet regarding this problem? I am at a loss as how to move forward. It seems like these problems were introduced only after we upgraded to the long read kits.

Cheers for the help.
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Old 06-22-2015, 05:38 AM   #10
mps4208
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The Agilent will not show the small reads which is frustrating, the only way to see them ahead of time is to actually do the 454 PCR "Sample Library Quality Control" in Tech bull #2011-002. I've pretty much decided that an extra AMPure XP clean-up is needed post library, we usually do two more 0.7 ratios (not following that bulletin-nor sizing solution). Also, don't "rinse" AMPure beads first, elute in 10uL using 10mM Tris pH 8.0.

It seems that because of the extra number of flows, it just exasperates the short read preference to a level so much beyond what we saw with regular length. I've noticed that amplicons are worse than gDNA. Amplicons I usually averaged 13-20K reads, which most customers said at the extra long length was still better for them than regular length with 100K reads.

Unfortunately, all this work for you may be for naught unless you have kits in the freezer. Roche 454 has just discontinued production of the XL kits for Junior at least.
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Old 06-23-2015, 08:06 PM   #11
darkhorse
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Default GS Jr+ kits out?

anyone experience same?
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Old 07-07-2015, 05:12 AM   #12
GSviral
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Hey guys,

Just a quick update.

Roche have released a bulletin regarding the XL+ kits. Many of them have failed Roche QC checks resulting in sequencing runs producing low pass filter rates and very low average read lengths. They have been recalled. Seems the standard short read kits are fine.
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