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Old 11-27-2012, 08:45 AM   #3
LVAndrews
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Location: Flagstaff, AZ

Join Date: Sep 2012
Posts: 55
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Hi Jim,

I too study environmental microbial samples. Coming from the world of JGI and large sequencing projects, I can imagine how you would be dissatisfied with the short reads offered by the leading NGS instruments at present. However, just because the tools we want aren't yet available to use doesn't mean we shouldn't learn what we can with what we already have. Carl Woese didn't even have Sanger sequencing at his disposal, and yet he changed the way we view life on Earth. I have used 454 and Illumina for analysis of fungal and bacterial/archaeal communities in my work. Each has its advantages and drawbacks, but they both provide far more information than tRFLP and clone library sequencing, and do it in a reasonable amount of time. Currently our lab uses a MiSeq for bacteria/archaea using the EMP protocol (http://www.earthmicrobiome.org/emp-standard-protocols/) and we hope to have a fungal assay in place soon. Our current workflow sequences a 250bp region of the 16S gene using 2x150 PE reads. Taxonomic assignments are made confidently, and this gives us our first real glimpse into the microbes that inhabit our various study sites. Additionally, knowledge of the presence of various species lets us design new testable hypotheses with regards to transcription of ecologically important gene products in the context of our established experiments.

Of course for metatranscriptome work you need a lot more depth to make confident assemblies of reads, but the tools we have will do the job for you right now. We've all been waiting for even a single word from Oxford for almost a year now, so the best strategy is probably to continue to embrace your functioning tools and adopt minions as necessary to augment existing data sets.

Andy
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