Curtobacterium flaccumfaciens (AKU)

Hi, this is a really long overdo post, but I figured it was about time to start blogging. My name is Jennifer and I am a third year Cell Biology and double in Communications. I am slightly new to the lab, jumping onto the Undergraduate Genome Project in late May. When I jumped on, I was posed with the task of rescuing the lost Curtobacterium from a myriad of petri dishes. After being sure we made a glycerol stock and running the 16S sequence through BLAST we identified the microbe to be Curtobacterium flaccumfaciens (which I will henceforth refer to as AKU), a gram positive soil bacteria and known plant pathogen. AKU could easily be identified as a phylogenetically informative microbe, because after checking RAST, we saw that neither Curtobacterium flaccumfaciens nor any other Curtobacterium species had been sequenced. However, everything afterwards proved to be far more difficult. After many failed library preps, particularly in the qPCR step (we weren’t getting enough active DNA) we decided that we would combine a TruSeq library and a Nextera library, hoping that the biases would be checked by each other. The bias mostly arose from the fact that we had to PCR 18 times for the Nextera library to get enough DNA which would definitely bias the reads for a microbe with as high of a GC content as AKU. We were eventually able to sequence both AKU and THP (Dietzia) with the new 250bp read MiSeq. However, we found that there was a large amount of E. Coli contamination an unfortunate side product of Nextera libraries. We were eventually able to throw out most of the E. Coli reads by lowering the stringency A5 uses to determine what is “trash” DNA. We also found that A5 can only accommodate 160bp reads so for now we are using a trimmer that cuts off 90bp so that we are at least able to run the assembly and not have it crash. We are hoping that we can somehow include those 90bp that we chopped off and even better be able to run A5 with 250bp reads. I will update you on more failures and triumphs in the near future!

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