Metagenomic with 454 Junior

[Pascale]

I try to sequence multiplex (21 samples) amplicons for months !! Amplicon size is around 609 pb (with MID ...). The problem is that the yield is very low (2%) with 2 or 4 cpb and when I sequence I juste have 50 bp instead of 609 pb (bioanalyzer is ok, no small fragments).

Do you think to increase elongation time of emPCR will be better or the problem come from an other step ?

Thank for your help !

[Aniki]

I try to sequence multiplex (21 samples) amplicons for months !! Amplicon size is around 609 pb (with MID ...). The problem is that the yield is very low (2%) with 2 or 4 cpb and when I sequence I juste have 50 bp instead of 609 pb (bioanalyzer is ok, no small fragments).

Do you think to increase elongation time of emPCR will be better or the problem come from an other step ?

Thank for your help !

You are experiencing things that a lot of users are experiencing. Do the following to minimize your losses:

1) increase your cpb to between 15 and 30cpb. I know this seems insane but it's the only way.
2) get a KAPA qPCR Lib-A/Lib-L kit to accurately quantify libraries.
3) ask for newest lots to be shipped to you and hope.

[RCJK]

You can also try the long amplicon emPCR protocol since yours is >600bp.

[Pascale]

Thank you very much for your advice ! I will try all of that this afternoom and I will let you know. Have a good day.

[Palecomic]

We size select twice with Ampure XP for amplicon sequencing, primer (and/or primer dimer) that is undetectable by bioanalyzer can still lead to a big short read spike. Our amplicon is 676 bp and the long emPCR protocol had an instant and amazing effect on improving the runs on our Jr.

Our optimized cbp is 0.35 - so seems very variable from site to site. The kapa kits are expensive, particularly relative to a Jr run, so we don't use them.