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454 emPCR failure mystery
Hello,
I’ve had problems with a 454 FLX run and nobody (neither Roche/454 or my sequence provider) is able to work out what is going wrong. The samples, which are amplicons of 200-300bp, appear to be of good quality, but the process falls over at the last hurdle - working on a small volume emPCR, but failing on the final large emPCR. Below is an outline of the scenario. I’d be very grateful for any advice, or to hear if anybody has had a similar problem (and hopefully solved it!).
Cheers,
I've initially amplified PCR products with M13-tailed primers, then attached LibL adapters via a second round of PCR (few cycles only). Running out on gel confirms the addition of the adapters. PCR products cleaned up with AgencourtXP, quantified with nanodrop (yes, low accuracy I know) and pooled. Pooled products were purified 2 more times with AgencourtXP.
• The pooled templates are of good quality and concentration, established by agilent bioanalyser, nanodrop and qPCR.
• There aren’t any short fragments that would cause problems. There are some fragments around 150bp, but they are within the expected size range.
• There are appropriate adaptors (LibL), established by qPCR and an emulsion titration.
• Small volume emPCR works well,
• Large volume emPCR gives much lower than expected results, and when we’ve tried sequencing the resulting beads they seem to cause failure of the processing filters, suggesting mixed reads.
This same scenario has played out for two separate projects - one metagenomic (single locus), and another that targets multiple loci in individual organism samples.
I'd be very grateful for anyone's thoughts.
I’ve had problems with a 454 FLX run and nobody (neither Roche/454 or my sequence provider) is able to work out what is going wrong. The samples, which are amplicons of 200-300bp, appear to be of good quality, but the process falls over at the last hurdle - working on a small volume emPCR, but failing on the final large emPCR. Below is an outline of the scenario. I’d be very grateful for any advice, or to hear if anybody has had a similar problem (and hopefully solved it!).
Cheers,
I've initially amplified PCR products with M13-tailed primers, then attached LibL adapters via a second round of PCR (few cycles only). Running out on gel confirms the addition of the adapters. PCR products cleaned up with AgencourtXP, quantified with nanodrop (yes, low accuracy I know) and pooled. Pooled products were purified 2 more times with AgencourtXP.
• The pooled templates are of good quality and concentration, established by agilent bioanalyser, nanodrop and qPCR.
• There aren’t any short fragments that would cause problems. There are some fragments around 150bp, but they are within the expected size range.
• There are appropriate adaptors (LibL), established by qPCR and an emulsion titration.
• Small volume emPCR works well,
• Large volume emPCR gives much lower than expected results, and when we’ve tried sequencing the resulting beads they seem to cause failure of the processing filters, suggesting mixed reads.
This same scenario has played out for two separate projects - one metagenomic (single locus), and another that targets multiple loci in individual organism samples.
I'd be very grateful for anyone's thoughts.
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