[Encarnita]

Hi everyone,
I am a new user of seqanswers, and I have so many questions. But to start slowly, ChritianBourne, what do you mean about new adaptor QC and post emPCR QC? Is there any protocol update? I am a Junior user though.
cheers!
Encarnita

[TonyBrooks]

I've sequenced as high as 27% and saw our usual levels of mix reads increase by approximately 1-2% (~8% to ~10%), so it's not devastating to sequence more.

We've also modified the protocol though to do three NaOH elutions as we never seem to recover all the bound capture beads from the enrichment beads with the recommended two elutions. This gives us a higher % enrichment than we would normally see if we followed the protocol to the letter - so that 27% may be a bit artificial.

[ChristianBourne]

Hi Encarnita,
I will impart my knowledge in return for a fully paid for holiday to Tokyo...! ;-)
The adaptor QC is a simple pcr using Lib-L or Lib-A primers and your prepared library to see if there's an acceptable level of amplification. Difficult to ascertain, but all you are looking for is an 'increase', depending on the quantity of your pcr reaction your sample will be diluted accordingly.
You need to check on a Bionalayzer, before and after, but of course if you diluted your sample down 1:50 (pcr reaction volume 50ul) then you wouldn't see anything, but you should see an increase! This is why it's difficult to measure and use accurately as a method of quantifying adaptor ligation success.
If your sample shows amplification then obviously the adaptor is there, but it won't tell you the % of library with the adaptor, this is a qualitative rather than quantitative test at present, I am working on the quantity of input DNA to make it more robust. You will know how off this is after your enrichment, too low at the correct calculation for copies per bead and what you normally use then you know the % is low, but that some of your library at least has the adaptor and will work after a new emPCR.
I've attached the protocol for "Procedure for the evaluation of amplification after emPCR".
This takes the first "melt' from enriching process, which is a ssDNA not required, off the capture bead and checked on an RNA Pico chip on the BA. It will show a mirror of the size of the fragments created in the emPCR and that will be subsequently sequenced on your capture beads.
Roche say the adaptor QC is better than the post emulsion QC, but have only put their name on the latter protocol.
We are pressuring them to certificate an adaptor QC protocol to standardise it for us all.

A word of warning, we introduced these extra QC after long discussions with Roche to make sure that what we were putting on was good sample, and that if they then failed it COULD be the machine (I'm aware samples may still have hairpins etc). We have had lots of samples which passed everything with flying colours but still failed.

[Anthony.287]

@ChristianBourne - Where did you get that protocol? I did something very similar two weeks ago, but I've never seen a protocol from Roche about it.

[ChristianBourne]

I got them from my FSA my friend.
It is my personal feeling that there is a lack of transparency from Roche regarding possible issues, rather than put out a bulletin telling all 454 users about issues. We've found out things from Twitter and Facebook and when we've asked they've tentatively confirmed it! Similarly I've been speaking to people at conferences etc and when they describe their issues I have to tell them what to look and why rather than them hearing from Roche! Not good customer service in that respect.

[Anthony.287]

Yeah, I can relate...
Any chance you would be willing to share your Facebook and Twitter sources? The larger this community, the better, I say. You can PM me, if you like.

[ChristianBourne]

We learned about calibration issues from Rachel Glover on twitter whilst our engineer was there funnily enough and there was a bit of confusion from Roche as to how the machine actually calibrates from the prewash file that is created pre run. My FSA is trying to find out what all the figures mean. As we had values that should have meant fluidics problems (sipper tube sitting flush with base of reagent tube perhaps...), but run was actually good, and vice versa!
The problems with reagent lot numbers are pretty common and sometimes it's just down to luck to find out from someone you know. That's why these forums are so useful

[Encarnita]

Hi ChristianBourne!
You are welcome to Tokyo as long as you don't mind baby's cry !
Thanks for your great answer and the pdf. I've heard about that QC, although I did not have any particular protocol, just the idea of neutralizing the DNA and analyse it by RNAChip. That's help though!
As for adaptor QC, I use DNA chip to check the quality and picogreen fluorescence to quantify. That need to have a adapted fluorometer though.

[ChristianBourne]

I'm just in the middle of doing some work to try and figure out what is the best way to check adaptor ligation with simple pcr without giving a massive primer dimer. You can obviously do a quick AmPure clean up but this may also get rid of small fragments that have been amplified that may also be amplified in the emPCR, so I'm playing with reducing the concentration of primer and with and without a clean up.
I'll keep you all informed.
Our 'control' run containing samples sent from 454 in America to run on our machines didn't work well. In fact considering there sample is what they use as a control my samples performed better!!!!
Wanna see the report...?
Sample details and enrichment values contained in word doc attached, 797A and 265_2 are my samples at two different concentrations of input DNA (half the amount so you'd expect half the enrichment, right...?), and pdf is the run report.
Enjoy :-)