I'm starting to get questions about sequencing progress, so I thought I would provide an update. I just spoke with our sequencing contact, and there have been some difficulties. I am on standby to order the sequencing kit for the instrument, but for the time being we have passed the last major hurdle in the process -- amplification.
A quick process review can be found in this blog post here.
The amplification process is done in a small tube with clear liquid. When the amplification process is finished you end up with a specific gene amplified a billion fold. However, it's still a small tube with clear liquid. In order to visualize if the amplification worked, we traditionally run it on a gel (agarose). The DNA is pulled through the gel, and the fragments separate out by size-- the smaller the fragment the faster it can move through the gel. This will result in it being pulled towards the "bottom" of gel faster. See below.
A quick explanation of the image. Each column is a sample except for "A" which is the standard or a "ladder." Each band in the ladder is a known length of DNA, and allows us to tell the length of DNA we amplified. Each black band in the rest of the lanes is the gene of interest. If a lane has no band, it means that nothing amplified. Most of our samples amplified, which I was really worried about. This is the last step in which individual sample properties could cause problems, so I consider it the last major hurdle. The sequencing itself is not trivial, but any issues at this point are purely technical.
We can see both Bacterial and Eukaryotic (fungi nematode etc..) DNA!
For this sequencing run, we are trying to capture the diversity of all life. You might notice in most samples you see a faint band above the thick black band. That band is actually the DNA from the fungi and other Eukaryotic organisms. Their gene (18S) is actually about 100 base pairs longer than the bacterial, 16S, gene thus it travels slower and separates out.
I've talked to a few people about bacteria and fungal ratios, and if we will be able to see nematodes and larger organisms. We will see something of fungi for sure, but this gel image really demonstrates one of the major problems with trying to look at all life in a single sequencing run. Mainly, there is just a lot more bacterial DNA... potentially orders of magnitude more depending on the system. Fungi and nematode cells are much much bigger than bacteria yet they both just have 1 set of genes per cell.
This is a problem, because we can only sequence a small % (50,000 out of billions?) of genes from each sample. Only a very small portion of those will be from larger organisms. Thus, we will unlikely be able to capture the true diversity of them or their true relative abundance to bacteria. To properly capture Eukaryotic diversity, bacteria should most likely be sequenced separately.
That's it for now, but get ready for a furry posts once we get some data to work with!
And any questions please ask away either by comment / email / phone.