Purpose:
The purpose of lab 2 was to listen and takes notes over all eight groups’ presentation over their assigned literature. As each group presented, they described the main purpose, challenges, and some of the advantages of their article. We then designed our own protocol for metabarcoding of soil ciliates so they could potentially be used during the lab in this semester.
Procedure:
- Turn in presentation on Canvas
- Present your article to the class
- Take notes over the remaining group’s articles
- Copy down all key notes and post on blog
- Listen and take notes over Dr. Adair’s presentation about the Ludox gradient centrifugation
- With your small group, design your own way to make a presentation Turin in the created protocol
Data and Observations
Group 1: we learned about cluster construction and operational taxonomic units
Group 2- Review article
Challenges: researchers had to be alert of random chimeric sequences
Application: numerous primers were research din this review article, we can use the results to determine which primers work best with ciliates and then experiment with multiple primers we can use databases a such as BLAST and MUSCLE to further research the primers that have been reviewed because of this research, we know that certain primers are better for testing certain groups than others Why use Chimeric Sequence? When doing the sequencing protocol, that are not real sequences. Chimeric sequence: part are form different organisms. You can tell by comparing sequences which ones are chimeric. They are artifacts.
Group 3- Primary article
Purpose: to detect zoonotic protists in raw sewage samples microbes pose as threats to environmental and public health similarities in DNA of the Avian microbial parasite to human microbes can indicate a possibility for the Avian microbial parasite to become zoonotic seen that in the past from swine flu
Challenges: field of genetic extraction is relatively new V4 and V9 have not been used to genetically distinguish very closely related species before this study Host DNA has the tendency to mask the microbial DNA
Application: The temperature at which the samples were stored at to best replicate the DNA extracted as 37C developed specific and effeccient timing cycles of heating and cooling to promote DNA replication Samples were grouped by extraction volume, primers used, and application region V4 worked better in taxonomy accuracy when compared to V9
Group 4- Barcoding Ciliates (primary)
Purpose: demonstrate how mitochondria cox1 gene can be used as taxonomic marker for barcoding and identifying Tetrahymena species Some conducted on 75 strains of Tetrahymena by Advantages present in all eukaryotic cells and functions homoguosly in a wide range of them more capable than 125 RNA and other mitochondrial genes easily amplified when using universal PCR primers less mutations because mitochondria reproduce via binary fission useful in distinguishing between closely related species
Challenges: The number of base pairs of the cox1 gene to amplify or obtain is still unknown
Application: cox1 gene can be used to speed up discovery of new ciliate species and provide new insights can be used by people who are not expert taxonomist to identify certain species
Group 5- Barcoding Eukaryotic Richness beyond the Animal, Plant and Fungal Kingdoms (review)
Purpose: discuss the advantages and limitations of DNA Barcoding
Challenges: multitude of genotypes that are hard to separate 18s rDNA is not effective enough to distinguish between inters the large majority of protists are currently uncultivable by known means or unavailable in culture Useful Information 18s rDNA is found in all Eukaryotes many copies/ highly expressed/ includes a combination of highly conserved and variable nucleotide sequences 2 step metabarcoding model Step 1: preliminary identification using eukaryotic barcode Step 2: group- specific barcode for ciliates Group 6- determine soil diversity (primary)
Purpose: Comparative analysis of the mitochondrial COI gene in ciliates
Challenges: COI gene can be used successfully in many animal taxa but not of all organisms incomplete knowledge of ciliate diversity and dispute exists on the estimates of Pertinent Research information COI gene is used to better discriminate between closely related taxa and can be used as a barcoding
Group 7- primary
Purpose: to test three different types of DNA separation methods finding the method that is the most efficient
Challenges: analyzing and interpreting the results certain extraction methods compromise DNA quantity for DNA quality different sites mean different results DNA isolation impaired by various factors
Application: Uniform method of extraction Low replication error extraction method
Group 8- Analyzing Articles on Metabarcoding (primary) Purpose: how the affects the biodiversity of the protists present in varying soil samples important to observe relationships with the environmental variables such as pH
Challenges: there is not enough information or past studies on solid protestant diversity that it is hard to compare the data application QIIME software was used to eliminate sequences shorter than 150bp to eliminate faulty segments
Adair Presentation
purpose: using density and an isopycnic centrifugation to sort particles in a particular medium that uses the same density as those particles. A density gradient centrifugation is used to separate very small molecules colloidal silica will be used with caution by wearing gloves relatively fast protocol
Group Design Protocol
Separation of DNA using the Ludox density centrifugation
Use the PCRs to amplify the DNA
Use the 13SrDNA sequence of DNA
Analyze the results using the QIIME software that aids in cutting out short sequences
Conclusion: Some of the articles that were presented were primary literature, while other, such as my group’s, were review articles. Both contained an abundance of information about the challenge that are encountered when using certain barcoding techniques and their specific purposes. Some of these techniques worked better in the area for distinguishing organisms that are relatively small, and some were effective in relatively large organisms. Our group designed a protocol that pulled from several techniques presented during the lab.
Future Steps
After my group designed our own protocol, it is now time to take future steps. Although we did not put much thought into our process, hopefully our method or someone’s in the class will be efficient enough to replicated and perform during lab this semester. After reading and listen to all the articles, I am fully prepared to encounter many challenges when facing barcoding.
