Drosophila 16S Paper: Difference between revisions
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Pretty self explanatory, but here is a table of all the libraries for which we have sequence data. The first column is the identifier that I will be using in all of my graphs and communications. | Pretty self explanatory, but here is a table of all the libraries for which we have sequence data. The first column is the identifier that I will be using in all of my graphs and communications. | ||
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Did we ever figure out why the libraries SCA and HPM have twice as many clones as all the others done at the same time? I have looked back at my notes and I SCA was one of the four original survey samples that was done first, so it is conceivable that it then went through again at a later date. | |||
= Example of Replication = | = Example of Replication = | ||
Revision as of 14:42, 16 April 2009
Technical Questions for Thought/Discussion
- how will Bellerophon handle incomplete sequences? Does the type of incompleteness matter, i.e. hole in the middle vs. only one half of the clone?
- So here is a graph of the number of unclassified clones in all of our laboratory samples. The overall question will be what do we do with these clones, and just looking at our lab samples is a good place to start. It is hard for me to imagine that this level of "unclassified" organisms results because they are actually novel or not found in greengenes. This is more likely a technical problem. As an example, DO and DM are both melanogaster on lab media, however the discrepancy between them is hardly trivial.
Overall, what we are to do with libraries like this, or with individual unclassified clones, is a question that I think needs to be resolved before we can get any credible information from the "classified" clones.
Scientific Questions for Thought/Discussion
Media:79_9Categories.pdf Here is a graph of all of our libraries (minus 3A, 3B, Ns and Wolbachia) at the genus level. The interesting thing to note is that taking all of our samples as a whole (lab/wild/interior/exterior/everything...), we get that a full 79% of the clones can be put into just 9 different categories. Although there is hardly a "core microbiome" since no genus is present in all the samples, it is cool to see that the same few things make up such a large percentage of our data.
This also brings up several technical questions. When we want to look at our data as a whole and make broad conclusions, which libraries should be included? There is the obvious distintions of lab/wild and interior/exterior/guts, but what about the different sequencing methods, sanger/phylochip, and the different DNA extraction methods. To answer the bigger question of just was a typical fly communities is composed of, I want to use as much data as possible. But many of our libraries (and not to even mention the corby-harris and Cox-Gilmore data) are slightly different from each other. Media:74_7Genera.pdf Here is the same as above, except that I have removed ALL the unclassified clones from the analysis. Note that now some of the libraries (Dml_CANS, DmW_Turr1/2, IMH) are now mainly composed of those genera not found in all the other libraries. We need to resolve if these differences are due to actual biology or just technical difficulties. But it still good to see that only a few genera make up a substantial portion of the dataset.
Lab Flies only: Internal vs External, unclassifieds still included-Media:intVSext+unC.pdf
Lab Flies Only: Internal vs External, gammaproteo and enterobacter unclassieds removed-Media:intVSext-unC.pdf Only D. melanogaster is represented in the above two files. The "grand total" column is the average of all the internal samples. The "external" column is the average of 4 external and media samples taken from both the Kimbrell and the Kopp labs, and hopefully is a good representation of what the flies are exposed to in the lab. Things to note: It seems as though the only genus that is enriched inside the flies is Providencia. In fact, Acetobacter and Lactobacillus, the two genera that we have been focusing on most heavily, seem to make up a relatively small proportion of the internal community. Next, it is interesting how the lines from the Kimbrell lab (all those that begin with DmL) are mostly Shigella, Microbacterium and Variovorax, while the Kopp lab samples (all the others) do not have those three Genera at all.
Table of all Samples Sequenced
Media:SurveySequencedTable.xls
Pretty self explanatory, but here is a table of all the libraries for which we have sequence data. The first column is the identifier that I will be using in all of my graphs and communications.
- Did we ever figure out why the libraries SCA and HPM have twice as many clones as all the others done at the same time? I have looked back at my notes and I SCA was one of the four original survey samples that was done first, so it is conceivable that it then went through again at a later date.
Example of Replication
Here is the best example we have of either biological or technical replication with our samples. The first four samples (DmW) are melanogaster collected on Citrus Fruit at Michael's orchard in August 2007. They are all 10 pooled dissected guts. The first one was done with bead-beating, while the second one the bead-beating was omitted. The amplified DNA was then hybridized to a phylochip. The next two samples were done with bead-beating and cloning and sanger sequencing. HCF and ICF are from D. hydei and D. immigrans collected off of Citrus Fruit at Michael's Orchard (and another one close by) in May 2008. They are both guts and at least 5 individuals.
Things to Note: The two different DNA extraction methods yield roughly the same outcomes. I didn't upload the graph, but once wolbachia is removed, the relative amounts of each genera is pretty much the same. Next, nothing convincing can be seen by comparing the two sanger sequenced replicates to each other or by comparing them to the Phylochip data, since they each are about 80% wolbachia. The one good thing to take out of this may be that if we are concerned that a population is infected with wolbachia, the bead-beating step could be skipped and this may not drastically change the results.
I included the ICF and HCF samples on this graph because they are the only examples we have of returning to a location in a different year and sampling on the same substrate. Unfortunately, both samples are dominated by unclassified clones. This may mean something biological (novel bacteria!) or could just be a technical artifact. Either way, it seems clear that the most prevalent genus in melanogaster from the year before (Acetobacter), is not present in these two samples.
Finally, check out the Mesorhizobium! I can't say that I was expected to see 56 clones of that.
Outline
Summary/Abstract
Introduction
Materials and Methods
Results
Discussion
Current Status
I can edit too
Artyom Just testing - looks like I can edit?
Jenna <- click here to see what I'm up to!
