PCR

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drevie
Posts: 67
Joined: Sun Feb 04, 2007 10:23 pm
Location: California Lutheran University, Thousand Oaks, CA

PCR

Post by drevie » Fri Feb 14, 2014 12:04 am

We started working on our projects. One has two low quality regions about 100 bp apart. When they tried to make PCR primers, the shortest product would be 2.2 kb! We relaxed the settings a little, but no changes in the primers occurred. Should we relax the settings a lot (e.g., lower Tm, etc.), or is there any other solution?

wleung
Posts: 185
Joined: Sun Feb 04, 2007 7:41 pm
Location: Washington University in St. Louis

Re: PCR

Post by wleung » Fri Feb 14, 2014 12:59 am

As Chris mentioned in page 4 of the Sequence Improvement Protocol for GEP Hybrid Assembly Projects document, our initial assessment of the D. biarmipes projects suggest regions with low consensus quality are rare because of the high 454 and Illumina read coverage. The low consensus quality regions are usually found either at the ends of the projects (which can be ignored) or are found near gaps (which will be resolved when you design reactions to close the gap). Because of the high read coverage, multiple low quality regions that are in close proximity to each other could indicate a misassembly.

The GEP Hybrid Assembly Walkthrough contains an example that has a low quality region because of a gap. This gap is actually caused by a misassembly and the walkthrough illustrate the protocol that your students can use to resolve the misassembly.

I should also note that, unlike previous Sanger projects, resolving misassemblies is not one of the GEP goals for the hybrid assemblies sequence improvement projects. Consequently, I would recommend consulting with Chris or I on the low quality regions prior to designing PCR reactions.

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