[DNA_Dan]

See Application Brief # 001-2009 Entitled Unidirectional Sequencing of Amplicon Libraries Using the GS FLX TItanium emPCR Kits. (Lib-L) On the second page, Primer A and Primer B are for fusion primers. Just order these sequences up to (not including) the key pass sequence.

[boerm]

This might be a possibility, but the equilibrium favors dimer formation. If you want to test for this simply do an amplification on the final library and run it on a BA chip again. If those fragments are there you should pick them up.

YMMV on expired reagents. It just depends how precious your samples are. Other labs with unlimited budgets throw them out.

Dear DNA_DAN,

Thanks for your suggestions, do you have the sequence of the Lib-L primers to amplify this library?

Thanks,

Martin.

[DNA_Dan]

If it is not the enrichment beads causing the problem and not the emPCR reagents, could the problem of short reads be caused by the MID adaptors, that form dimers during the library preparation.

Can it be that these dimers are not visible in the bioanalyser because they are sticking to the larger DNA fragments?

Our MID adaptor kit is over the expire date, could this explain the problem?

This might be a possibility, but the equilibrium favors dimer formation. If you want to test for this simply do an amplification on the final library and run it on a BA chip again. If those fragments are there you should pick them up.

YMMV on expired reagents. It just depends how precious your samples are. Other labs with unlimited budgets throw them out.

[boerm]

If it is not the enrichment beads causing the problem and not the emPCR reagents, could the problem of short reads be caused by the MID adaptors, that form dimers during the library preparation.

Can it be that these dimers are not visible in the bioanalyser because they are sticking to the larger DNA fragments?

Our MID adaptor kit is over the expire date, could this explain the problem?

[bradfish]

I've been loading one emPCR at a time as I figured letting it sit would be an issue. I usually pause when you add the amplicon to the oil, and just move forward with one from there all the way through the procedure, then add amplicon to the next oil and start from there again. Seems to work great.

Interesting thoughts on diversity of pool correlating with signal interference. What I am are pools of many different samples each containing at least a few different organisms, many containing much more. I am going to look into this correlation when I get time.

[DNA_Dan]

Right now I don't have time to post a lot of data, but I can tell you we are doing 1 cpb with new and old lots. Sequencing 400 bp amplicon, with 1/4 the amount of reccomended amp primer. We went from getting 400-600k enrichement consistenly for the last year to over 1 million on the last few runs with nothing changing except the lots.

We are doing the same except using Lib-L kits on a FLX in LV format. Key is reducing that primer amount. Also I think if you are sequencing all the same amplicon, you have higher signal inteference and this gets filtered out in post processing as a short or failed read. The passing filter rate drops significantly the less unique the sequencing pool is.

As for the increasing enrichments with the newer kits, we haven't really noticed this, however our enrichments are higher on plates that sit for a while after the final emulsion shake. We find that these "false" higher enrichments tend to sequence poorly. Same CPB, Same reagents, just the 1st cup sitting out longer while the second is being aliquoted = poorer sequence. As a result, we have gone to processing one LV cup at a time, putting them on the thermalcycler ASAP once aliquoted. Processing the cups this way is much more consistent, albeit more time consuming.

[Aniki]

so....

I am having one heck of a time with old emPCR kits. I am having an enrichment nightmare where I simply cannot get any freaking enriched beads out of this (extremely low %, regardless of CPB). I hope these new kits can do better for me but you guys aren't exactly giving me confidence.

[GraemeFox]

I enquired to Roche about just how the concentration of the primer in the emPCR kits had been changed and I have just had a response.

"The concentration of the primer in the kit was actually deceased (sic) by about 4 fold."

[ECO]

Let's keep it relatively serious and professional, Dope. Thanks.

[adaptivegenome]

I don't know whether to laugh or cry about this post. And yes, you certainly are a big dope.

Posting garbage once is possibly funny, posting it more than once is spam.

[bradfish]

My group actually figured out that if you don't add any of the emPCR enzyme mix, we would get excellent sequencing data. We dun' get it but we keep on keepin' on ya know?

I don't know whether to laugh or cry about this post. And yes, you certainly are a big dope.

[bradfish]

Actually had a decent run yesterday, given the huge enrichment. 90k reads, only 88k short quality. Maybe it was a fluke? Running another tonight, this will sway me one way or another.

[Anthony.287]

I wonder if there are actually short fragments, or if it's one of the other parameters that is included in the short quality filter..I'm not familiar with the Jr, but this was a common problem with the FLX.

[bradfish]

So you've gone from ~10% enrichment to 20-30%. Interesting.

Thats true, but its a low quality enrichment. My overall passed filter reads have been reduced in half from 100k to 50k, because of short quality failures.

[Anthony.287]

So you've gone from ~10% enrichment to 20-30%. Interesting.