THE PROCESS OF SEQUENCING THE STARTERS AND POTENTIAL RESEARCH QUESTIONS
Now that we've finished our starters, they're in the process of being sequenced, and we know a little bit more about DNA sequencing, we're going to hop into what exactly we're sequencing and what kind of research questions we can answer with what we're sequencing.
SO, WHAT ARE WE SEQUENCING EXACTLY?
We're sequencing specific regions of the genomic DNA, called the 16S rRNA, which is typically used when sequencing bacteria, and the ITS region, which is typically used when sequencing fungi and other eukaryotic organisms. The method by which we're only sequencing these two regions is referred to as amplicon-based metagenomic sequencing (where amplicons are just little pieces of the DNA).
If we had sequenced the entirety of the DNA of the starters, then we would've performed shotgun metagenomic sequencing, which leads to wondering, why did we choose the method we did to sequence our DNA?
THE ENTIRE DNA VS. PARTS OF THE DNA
Now, I have a few theories as to why one would choose shotgun metagenomic sequencing in some scenarios and amplicon-metagenomic sequencing in others. To understand this, I first had to interpret the pros and cons of both methods, starting with amplicon-metagenomic sequencing.
Some of the major pros I interpreted amplicon-metagenomic sequencing to have are that they focus on one region of the DNA, which means its easier to find the similarities and differences between organisms that are similar to each other. For instance, all the fruit starters might have similar DNA, meaning looking at one region is better in the grand scheme of things because we are able to better point out key similarities and differences.
However, one of the major cons I found for amplicon-based metagenomic sequencing was that it is a very time-consuming process. When one sequences the entire DNA, they don't have to do any extracting, meaning its much less time consuming, but when one sequences certain portions of the DNA, they have to extract the specific amplicons of the DNA they want, which adds more time to the sequencing process. This method ultimately seems easier in terms of analyzing the data at the end, but more difficult in terms of preparing the sample before it can be sequenced..
Amplicon-metagenomic sequencing - Above is an infographic describing how amplicon-metagenomic sequencing is carried out.
With that being said, one of the major pros of shotgun metagenomic sequencing is that it is much less time consuming because, once again, we don't have to do any extracting.
While its much less time consuming, one of the major cons of shotgun metagenomic sequencing is that it can be harder to focus in on the key differences and similarities of similar organisms. For instance, the fruits that we used in our experiment might have very similar DNA, meaning it'll be harder to compare their similarities and differences since they will all probably look very similar. Sequencing the entire DNA means we also aren't focusing in on the regions of DNA in our different microbial communities that are responsible for causing rising and the formation of yeast, which makes the answers we're searching for a whole lot harder to find. This method ultimately seems easier in terms of preparing the sample before it can be sequenced, but more difficult in terms of analyzing the data at the end.
After being able to better understand the pros and cons of the two different methods, I think I have a better understanding as to why we chose the amplicon-metagenomic sequencing question. See, the metagenomic sequencing method will help us better answer questions, such as what microbial species cause yeast to rise, compared to the shotgun metagenomic method. Since our yeasts are most likely very similar due to fact that they're all fruit based starters, focusing in on specific regions helps us to better understand what differing factors cause our yeasts to rise/not rise at different rates.
While it seems the amplicon-metagenomic sequencing method allows us to answer more specific questions, shotgun metagenomic sequencing seems like it'd only be able to help us answer way more general questions or questions about completely differing organisms. Basically, amplicon-metagenomic sequencing teaches us more than shotgun metagenomic sequencing because it helps us understand specific, KEY PORTIONS of the DNA in a more detailed light.
Now, that we have a better understanding as to why we used amplicon-metagenomic sequencing in this experiment, I have a few potential research questions that I feel we might be able to answer with our sequencing data.
POTENTIAL RESEARCH QUESTIONS
Three potential questions I thought could be answered by the data set are:
1.) Do "thicker/starters with medium thickness" banana starters have different microbial compositions than "thinner" banana starters? Basically, I want to see if the thickness of starters affects the types of microbes or ratio of microbes present in our different starters.
2.) Do starters with fruits classified as "bitter", have rising rates that differ from fruits that are classified as "sweet"? With this question, I want to explore if fruits that have differing tastes have microbial communities with differing compositions.
3.) Do fruits with similar rising rates have microbial compositions that are similar/the same to other fruits with a similar rising rate? Finally, I wanted to explore if fruits with differing rising rates had varying microbial compositions.
TO BE CONTINUED...
In conclusion, amplicon-metagenomic sequencing is a way to examine parts of the DNA, whereas shotgun metagenomic sequencing is a way to examine the entire DNA sequence. With this being said, examining portions of the DNA makes it easier when we are examining data after sequencing, but examining the entire DNA make it easier when we are preparing our DNA for sequencing. Overall, amplicon-metagenomic sequencing is better for our experiment because it helps us to explore our questions, such as 'what are the differing microbes that seem to play a role in how our starters rise?', in much more detail than if we had sequenced the entire DNA.
REFERENCES:
https://www.lcsciences.com/documents/sample_data/metagenomics/Metagenomics_html_report_DEMO.html
Great questions! Do you think we have enough data to answer number 1? If not, what experiments would we need to do?
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