Micrococcus luteus wasn’t interesting enough to warrant further analysis, so I have picked another organism, TDU, to begin constructing a genomic library of. It appears to be within the Microbacterium genus, and shows identical similarity with the species oxydans. We actually isolated several Microbacterium colonies throughout this project from different sources, so I had a number of samples of which I could choose from to begin moving forward with.
A phylogeny of the different Microbacterium samples we isolated was built by David Coil, to help me visualize how similar the samples are to the published genome. There is one completed and published genome in the Microbacterium genus, for the species testaceum, so the goal of the tree was to help me pick from the most divergent organisms to minimize the chances of a duplicate publication of the same organism’s genome. This tree shows the comparative similarities of the Microbacterium species we found with the published Microbacterium testaceum genome recovered, with the most divergent organisms appearing to the left. UPDATE: Two outgroups have been added to further illustrate the degree of divergence
M. testaceum 16S sequence_alignment_tree
The three samples I picked as the best candidates were AV2, TDU, and TFU (TJU was a difficult and sloppy process to isolate, so I played it safe and avoided it altogether). AV2 had very, very low concentrations of DNA in the genomic preparation, so the sample was discarded. TDU and TFU both contained high levels of genomic DNA in their genomic preparations, so both were still equally viable as candidates. When I checked the glycerol stocks of both organisms on plates however, TFU appeared to have slight contamination (which is really bad, considering these stocks are our last resource for obtaining pure samples of these organisms). This confirmed TDU as the Microbacterium oxydans sample that I will begin working with to construct a library of.
Currently, the dilution streak of TDU is incubating at 37 degrees C, and tomorrow I will begin the process of confirming the glycerol stock and begin the tagmentation reactions for the genomic library.
Jonathan Eisen's Lab 
Thanks for the additional detail
Some questions
1. What does “identical similarity with the species oxydans” mean? Can you clarify?
2. Can you explain more what is in this tree and what it shows? What do the numbers mean? How were organisms/sequences selected to be put on the tree? How was the tree made?
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1. I didn’t use the best choice of words there, admittedly. Both TFU and TDU had ribosomal sequences that were 100% identical with many Microbacterium oxydans ribosomal sequences on BLAST, and no other species were among the top hits. This lead me to conclude that both of these organisms are most likely M. oxydans
2. The horizontal distance on the tree represents the degree of divergence from the published Microbacterium genome (it was supposed to be labeled m. testaceum but the program cut off part of the name). The numbers at each node represent the bootstrap value of that node, which measures the degree of certainty of the relative branching order for the phylogram. For example, we know M. testaceum is the sister taxa to TAU and TCU with 100% certainty, but we are only 83.1% sure that TAU and TCU are sister to each other.
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I should note that when I say the taxa are “sisters” I mean purely in the context of the phylogram that David constructed. In practical terms, this means we know that TAU and TCU are more or less identical to each other, and they both are very similar to M. testaceum. The other samples are much more divergent relative to TAU and TCU, but are all very similar to each other.
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I note- the tree is relatively hard to interpret b/c there is not an outgroup in it .. it would be good to have some other taxa more distantly related which would help show the relative “closeness” of these samples to each other.
I also note that this is just rRNA genes. Many strains that have identical rRNA genes to each other have major differences in their genomes.
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I replaced the older tree with a new phylogeny that includes two outgroups, Clavibacter michiganensis and Tropheryma whippelii, to show the divergence of the other Microbacterium species relative to to these two organisms, which fall under different genera than Microbacterium testaceum (and thus should be more divergent than any other Microbacterium species)
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Thanks
Question: how was the tree made?
Question: what is the meaning of the branch lengths and #s?
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16S consensus sequences were generated using CLC Genomics Workbench, and these sequences were then exported to the program Dendroscope. This program created 100 hypothetical phylogenies based on the consensus ribosomal sequences, and produced this tree as the most likely based on these simulations. The numbers at each node represent the number of times out of 100 in which that particular branching order occurred. The horizontal lengths of the branches represent the degree of divergence from the node
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