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Sequencing Update-- Almost there!

Hey Everyone,


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.


cell size comparison: bacetria fungi virus human hair
https://askabiologist.asu.edu/explore/puzzling-pathogens

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.






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8 Kommentare


Zack Jones
Zack Jones
04. März 2021

Yes that is correct, I am using the primer set that can amplify both. It is also now considered the best primer set even for just 16S alone. The 18S par to this study is purely experimental (grant was also written 2 years ago now) and yes it is kind of "free." I am not very familiar with Eukaryotic sequencing of 18S. It is still in early development and even if you amplified it and sequenced it separately I have no idea how good the databases would be to identify the sequences. In my opinion, microscopic Eukaryotic life like fungus/nematodes/ ameboa/ diatoms are the least understood form of life right now. We still mostly know about them by looking at…


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Zack Jones
Zack Jones
07. März 2021
Antwort an

Oh fantastic!I will definitely look more into it and chat with Gary about it since he is more up on this stuff than I am. I think I last heard they were working on putting more cost into the instrument to make consumables much cheaper. All the sequencing tech is pretty crazy but the MinION pore sequencing is especially nuts. https://nanoporetech.com/how-it-works *some dramatization added* It actually reports a unique signal for every 3 base pairs but 1 moves basepair at a time so you actually need like a semi- artificial intelligence program to call to bases which can take more time than the actual sequencing. I am really excited about the possibility of full length 16S sequences (~1550bp) as i…

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rachel.kulberg
rachel.kulberg
03. März 2021

Ok so you are using the 16S primer that has this mutation so that you can amplify both fungal and 18S genes together in a single sample to save on costs and time ?

Once you amplify the genes, you send them out for sequencing? Have you looked into buying a NGS instrument- you can tag primers allowing separate genes to be identified in a given sample or test multiple samples in one sequencing run- the costs of these instruments are surprisingly affordable and each run is inexpensive as well. BTW, is there high sequence similarity between bacterial 16S rRNA genes and if so, what bacterial species can be separately identified ? Is this true for different fungal species? (If…

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Zack Jones
Zack Jones
03. März 2021

Hi Rachel!


Yes this is typically how sequencing was/is done. Bacteria and Archaea are targeted using 16S primers. This is how microbial sequencing started mainly because the 16S gene was the first to be identified as a "good" biomarker gene. Fungi were then able to to be sequenced and identified by targeting the ITS gene. This is also still commonly done. Fungi and other organisms can also be targeted using the 18S gene which I believe is still less common and still pretty experimental. Also just because you can amplify the gene doesnt mean you can identify it in a database... whole other problem especially for new biomarker genes. The 16S and 18S gene are pretty much the same gene, just…


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rachel.kulberg
rachel.kulberg
03. März 2021

A bit confused- can you use specific primers for bacterial species verses specific primers for fungal species for the PCR reaction? If so I’m interested in which genes are the best to target to distinguish between these various species ?

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