Chandler Opara 

April 25, 2019

Poster Presentation and Abstract

Objective/Purpose:

The objectives of the lab were to vote on a logo, complete our final abstract, and make the finishing touches to our poster. The purpose of completing the abstract and poster is to be able to present  it in the Symposium.

Procedures:

1. Vote on logo design presented in the PowerPoint .
2. Input all the metadata in the class Excel spreadsheet. Label the sample with the criteria stated in the spreadsheet.
3. Finally, use the critiques from the rubric to revise and finalize the poster. Write out the abstract and edit the poster title if necessary. When finished, upload the poster to the Box and Canvas.

Data:

Abstract

Ciliates play a major role in the food web which impacts all hierarchies of life. There is vast biodiversity in the rhizosphere of Earth, containing ciliates and other microorganisms (1). Little is known about the abundance of organisms that reside in the soil of terrestrial environments. This study was conducted to determine the diversity of soil ciliates in the rhizosphere on Baylor University’s campus. The process included collection, extraction, and amplification of ciliate DNA from the soil samples (2). The sample was retrieved from a Quercus Virginiana tree on Baylor’s campus in sandy loam soil. Our PCR for the 18S V4 primer resulted in a positive DNA band although the DNA concentration was not completely pure. Our results indicate that there is a large concentration of ciliates in the rhizosphere of trees on Baylor University’s campus.

Storage:

Tubes were placed back in the rack after use.

Conclusion/Future Steps:

In conclusion, we were able to revise our poster based off of the critiques, draw better conclusions from our results, and fix any minor details. The next step would be to present our posters at the symposium on May 2.

Chandler Opara

April 18,2019

 Soil eDNA Metabarcoding Analysis: QIIME 2

Objectives/Purpose:

The objective of the lab was to analyze the Chelex eDNA sample and PowerSoil eDNA sample using QIIME2. The purpose of doing this was to examine the presence of the diverse ciliate species.

Procedures:

Re-Installing QIIME2:

1. Go to the Miniconda website and download 64-bit (.pkg installer) version of Miniconda Python 3 for Mac OS X computers.
2. Using the launch pad, open up the Terminal and type conda update conda. Type y to start the downloading process. Make sure all the files have been downloaded.
3. Copy and paste the link from the webpage called “Natively installing QIIME 2” into the command of the Terminal to start the cleanup process.
4. Activate the QIIME 2 environment by typing “source activate qiime2-2019.1” into the command
5. In order to test the installation, type qiime –help in the terminal. Check if there are any errors when running the command.

Soil Edna Analysis with QIIME2

1. Go to the link BOX Folder and download the folder with the files. The following files are Emp folder, Qiime walkthrough, sample metadata, and silva
2. We will use the Qiime walkthrough document to complete the analysis.
3. Open a neew terminal and activate Qiime2 by typing:

• source activate Qiime2-2019.1

4. Next, create a folder named CILICURE_2018. In the terminal, change the directory by typing in:
   • cd CILI-CURE_2018
5. Import sequences as QIIME2 artifact:

• qiime tools import \–type EMPPairedEndSequences \

–input-path emp-paired-end-sequences \

–output-path emp-paired-end-sequences.qza

6. The next step would be to demultiplex the sequences. This means that the sequences will be grouped according to the samples they belong along. In order to do this, type the following:

• qiime demux emp-paired \–m-barcodes-file sample-metadata.tsv \

–m-barcodes-column BarcodeSequence \

–i-seqs emp-paired-end-sequences.qza \

–o-per-sample-sequences demux.qza \

• q qiime demux summarize \

–i-data demux.qza \

–o-visualization demux.qzv

7. The .qza file was converted into a .qzv file for viewing.To view this qzv file, open the webpage “QIIME2 View” on your browser. Drag the qzv file into the chosen area in the webpage. This showed a summary of our demultiplexed results.
8. After demultiplexing the sequences, we need to denoise the sequences. This mean we are just cleaning up any messy sequence data. We denoised the sequences using DADA2 to 220 bases. In order to do this, input the command:

• qiime dada2 denoise-paired \

–i-demultiplexed-seqs demux.qza \

–p-trunc-len-f 220 \

–p-trunc-len-r 220 \

–o-table table.qza \

–o-representative-sequences rep-seqs.qza \

–o-denoising-stats denoising-stats.qza

9. A feature table was created to summarize all of the results. To create a feature table, input the command:

• qiime feature-table summarize \

–i-table table.qza \

–o-visualization table.qzv \

–m-sample-metadata-file sample-metadata.tsv

• qiime feature-table tabulate-seqs \

–i-data rep-seqs.qza \

–o-visualization rep-seqs.qzv

• q qiime metadata tabulate \

–m-input-file denoising-stats.qza \

–o-visualization denoising-stats.qzv

10. The .qza file was converted into a .qzv file for viewing.To view this qzv file, open the webpage “QIIME2 View” on your browser. Drag the qzv file into the chosen area in the webpage. This will show the feature table.
11. After creating a feature table, we created a phylogenic tree by inputting the command:

• qiime phylogeny align-to-tree-mafft-fasttree \

–i-sequences rep-seqs.qza \

–o-alignment aligned-rep-seqs.qza \

–o-masked-alignment masked-aligned-rep-seqs.qza \

–o-tree unrooted-tree.qza \

–o-rooted-tree rooted-tree.qza

12. Finally, we created a chart with taxonomic classification to explore the taxonomic composition of the samples, input this command:

• qiime feature-classifier classify-sklearn

–i-classifier silva-132-99-515-806-nb-classifier.qza

–i-reads rep-seqs.qza

–o-classification taxonomy.qza

• qiime metadata tabulate \

–m-input-file taxonomy.qza \

–o-visualization taxonomy.qzv

• qiime taxa barplot \

–i-table table.qza \

–i-taxonomy taxonomy.qza \

–m-metadata-file sample-metadata.tsv \

–o-visualization taxa-bar-plots.qzv

13. In order to view this qzv file and taxa-bar-plots.qzv file, repeat step 10.

 Data:

 Demultiplexed Results:

Denoise Results

Taxonomy Bar Plot Results

Conclusion/ Future Goals:

In conclusion, this lab allowed us to create a visual representation of the biodiversity found in our sample. The taxa bar plot showed that the PowerSoil sample contained around 25% of organisms from the supergroup SAR when compared to the Chelex sample which contained 54% of those organisms. The visualization and analysis of the data helped provide information about the community composition and added information to a scientific database, which connects similar research to other resources. With the use of PubMeD, we were able to see the various identifications of the ciliates. In the future, I am curious to see how the CyVerse platform and Jupyter Notebooks will provide more information about our sequences. Overall, this lab was successful since I have become more comfortable with the QIIME2 program and using the terminal.

Chandler Opara

April 4, 2019

QIIME 2 Installation and Terminal Practice

Objectives:

The objective of this lab was to use the Apple Mac OSX terminal to install the QIIME program. The purpose of this lab was to familiarize ourselves with the different type of codes used in the terminal. This program will allow use analyze the data collected from our samples in the following weeks.

Procedures

Installing QIIME 2

1. Open Terminal on Mac by using the launch pad
2. Practice using the terminal by stating commands
3. Download Miniconda(64-bit (pkg installer) ) to begin the process of download qiime
4. Close terminal and then reopen the terminal
5. Once downloaded, type “conda update conda” into the command
6. Type “y” to proceed with the process
7. Type “conda install wget” into the command
8. Type “y” to proceed with the process
9. Copy and paste the link from the webpage called “Natively installing QIIME 2” into the command
10. Activate the QIIME 2 by typing “activate qiime2-2019.1” into the command
11. Type: qiime –help in the terminal to test the installation

Data

Common Command Used

“Is”: list applications

“cd~/Documents”: to change the directory

“mv” command : can be used to move a  file

Conclusion/Future Goals:

In this lab, we were becoming familiar with the QIIME 2 Program to help us better understand how to analyze the data given to us. Understanding the analysis of our data will help us determine the various kinds of microorganisms within the samples. This will allow us to gather more knowledge about the diversity of the community and conclude how the eDNA impact the surrounding area. In the future, I’m curious to see what new information the reads will give us and how we will interpret them. I’m also excited to see how the use of QIIME 2 will contribute to the conclusions being made.

Chandler Opara

March 28,2019

Next Generation Sequencing and Cloud Computing 

Objective/Purpose:

The objectives of this lab were for the students to learn about Next Generation Sequencing (NGS) and be introduced to CyVerse, the computing program.

Procedures:

1. A PowerPoint about the steps of Illumina sequencing was viewed and discussed.
2. Work with your assigned partner to arrange the Illumina Cards in the correct order.
3. After checking with the LA, the QTM was filled out to fully comprehend the procedure of NGS.
4. Students put their CyVerse usernames in a spreadsheet.

Data

Conclusion and Future Goals:

In this lab, my partner and I worked together to understand the concept of how the process of DNA sequencing works. We learned about the role of the adapter, tagmentation, bridge amplification, and clusters. This knowledge will help us determine how we should identify the types of species in our samples. The future goal is to develop a protocol that will allow future students to sequence DNA and learn more about the diversity of the soil ciliates. In the future, I hope to get more comfortable with the cloud computing software because as of right now, I do not fully understand it.

Chandler Opara

March 21,2019

Lab 9: Poster Presentations

Objectives: The objective of the lab was to present the rough draft of our posters that we made from the previous lab. The purpose of this was to receive helpful critiques to improve our posters. Also, this allowed us to see how other groups used different elements and techniques and compare what parts did well and what needed adjustment.

Procedures:

1. Present poster presentations
2. Receive feedback from Dr.Adair
3. Grade other group’s presentations
4. Using the given feedback, evaluate and make changes to improve our poster.

Critiques:

1. Fix and crop the figures
2. Add the tree metadata
3. Include references gathered from the articles used
4. Put references and acknowledgements in the same area
5. Label the tree picture
6. Insert the agarose gel amount in the data captions
7. Edit the introduction

Updated Poster Design:

Conclusion/Future Steps:

In conclusion, this lab allowed us to see the improvements that need to be made to our posters. This helped me see what a good quality scientific poster should be composed of.  In the future, we will work on making changes to our poster, so the final draft of the poster will be a good balance of important information and figures.

Chandler Opara

3/8/19

PCR Results and Scientific Poster Design 

Objective/Purpose:

The objective of this lab was to analyze our PCR samples through gel electrophoresis to determine if the samples were able to be sent off to sequencing. We also took our midterm quiz and  provided a rough draft or our scientific poster.

Procedures

Gel Electrophoresis

1. The 1.5% gel was previously prepared.
2. Pipette 5 µl of Ladder into the #1 well
3. Pipette 10µl of the DNA sample into well #2 of the gel
4. Pipette 10µl of the control sample into well #3of the gel
5. The other group’s control and DNA sample were placed in the next two wells.
6. Run the gel at 100V for 30 minutes.
7. Use the imaging software to view the results of gel.

Data:

Storage:

Dispose of micropipette tips and gloves in designated area.  The control and treatment samples were placed back in the ice box.

Conclusion and Future Steps:

In conclusion, we analyzed the DNA samples to see if they are applicable to perform PCR and have the purity to be sent off for sequencing. From the use of gel electrophoresis, we were able to see the bands of the DNA in our treatment sample. This shows that we have a good quality DNA sample that can be amplified and sequenced. In the future, we will see the results of our sequenced DNA and we can interpret it to see how it relates to the soil diversity in our tree.

Chandler Opara 

2/28/19

Objective/ Purpose:

The objectives of the lab were to setup a PCR reaction using our eDNA sample and begin working on poster design layout. The purpose of making a PCR reaction is to amplify the DNA using V4 Ribosomal primers

Procedures

1. Using the result of the purified DNA sample concentration, calculate the amount of DNA template needed to make a total volume of 25µl.
2. Dilute DNA sample if needed (we didn’t need to)
3. To find the amount of sterile water needed, add the total amount of 2X Master, DNA template, and primers together and subtract from 25µl total volume.
4. In order to create an aseptic environment, clean the lab area with 10% bleach and wear gloves while performing the experiment.
5. Obtain an ice bath and place an empty tube, water, DNA sample, and V4 Primer in it.
6. Obtain two tubes which already contain 12.5µl 2X Taq Mix. One tube is the control, while the other is the treatment.
7. Add 1µl primer and 11.5µl water in the control tube. Centrifuge for 15 seconds.
8. Add 1µl primer, 1µl DNA template, and 10.5 µl water in the treatment tube. Centrifuge for 15 seconds.
9. Label the tubes. Place the tubes in rack with the other samples and label the location of the tube on a sheet of paper.

Data

Storage

The micropipettor tips were disposed in the designated cup. The ice bath and its content were returned to the correct area. The DNA tubes were placed in rack and labeled with its location. The work area was cleaned before leaving the lab.

Conclusion and Future Steps

In conclusion, this lab went well due to the fact that we were able to create the DNA tubes easily. Our group didn’t have to dilute our DNA sample since our concentration was 96.3ng, which is very close to 100ng. I am curious to see what kind of results the PCR will bring and how we will interpret them. For the poster design, I am going to look over the different layouts of scientific posters to get an idea for our poster design. Also, I am going to start preparing for the midterm exam.

Chandler Opara 

2/21/19

Objectives

The objectives of this lab were to develop a protocol for our experiment on ciliate diversity, run gel electrophoresis, and use a nandrop spectrophotometer to determine the concentration of DNA in our sample.

Purpose

The purpose of using running gel electrophoresis was to determine if any DNA and analyze how much of it was in our samples. We used the nandrop spectrophotometer to examine the amount of DNA concentration in our samples. The imaging software technology helped captured an image of DNA bands within our gel.

Procedures

Gel Electrophoresis

1. First begin practicing the technique of pipetting the loading buffer in the gel with Dr. Adair. When you do this on your own, wear gloves to prevent contact with Ethidium Bromide.
2. Remove the gel from the mold and place it in the electrophoresis chamber with the wells at the negative end of the chamber.
3. Pour 1X TAE buffer over the gel and the chamber.
4. After assigning which well will contain each DNA mass standard and sample, draw a picture to illustrate this diagram.
5. Extract 9µl of DNA sample and transfer it into the PCR tube and add 1 µl of 10X loading buffer.
6. Centrifuge the tube for a short time.
7. Add 10 µl of DNA into the 7^th
8. Pipette 5 µl of small DNA Mass Standard into one well.
9. Pipette 5 µl of high DNA Mass Standard into one well.
10. Add 10 µl of DNA of the other’s group sample into the 2^nd
11. Run the agarose gel at 100V for 20 minutes.
12. Take the gel to the lab, place it on UV tray, and run the imaging software to see the DNA bands.

Nandrop

13. Make sure the machine is clean and dry. Neutralize the machine by pacing a drop of water on the machine and running it.
14. Place 2µl of DNA sample on the top of the holder and close the machine to let it run.
15. Record the purity of DNA sample.

Data

ng/µl: 96.3

A260/A280: 1.61

A260/230: 0.47

Storage

The chamber was bleached, cleaned, and put in the correct area. Other equipment were cleaned and stored. Our work station was cleaned and wiped down.

Conclusion and Future Steps

In conclusion, we were able to see the DNA in our sample from the use of gel electrophoresis. The results showed us the different sizes of the DNA bands compared to our DNA mass STDs. The Nandrop Spectrophotometer analyzed the concentration and purity of the DNA in our sample. I was glad that our group’s DNA purity was close to the standard purity of DNA. The purity of DNA is 1.8, while our DNA’s purity is 1.61, which was very close. This revealed that were some impurities in our DNA. In the future, we will comparing our DNA bands to others and gather more information to complete the research paper.

Chandler Opara 

2/14/19

Objective

The objectives of this lab were to purify the DNA extracted from our soil samples and create an agarose gel in gel electrophoresis.

Purpose

The purpose of his lab was to complete the final steps of DNA extraction and build on our knowledge of gel electrophoresis by creating the gel.

Procedures

DNA Purification

1. The “crude soil DNA” extraction was brought up to 1 ml using sterile water. This 1 ml was added to a 15 ml tube.
2. 2 ml of warm DNA resin was added to the tube.
3. A column bottom and syringe barrel was assembled and put on a vacuum filtration.
4. All of the DNA resin was added to the column and the vacuum was turned on until all of it was filtered through.
5. After this was done, 2 ml of 80% isopropanol was added in 6 increments.
6. The column was removed and placed in a 1.5 ml tube and centrifuged for 5 minutes.
7. The column was removed from the tube and placed into an 80℃ heat block for 30 seconds-1 minute.
8. The column was the placed in a new 1.5 ml tube and 50 µl of sterile water (heated to 80℃) was applied directly to the column.
9. The column was incubated for one minute and then centrifuged for another minute.
10. The tube was labeled and placed into the freezer.

Agarose Gel

1. Added 4mL of 10x Tris-EDTA-Acetic acid stock (TAE) and 36 ml of DI water into a conical tube to create the stock solution.
2. Weigh out 0.4g of agar and placed it into an Erlenmeyer flask.
3. The 40 ml of the stock solution was added to the Erlenmeyer flask.
4. The flask was heated in the microwave for about 1 minute and gently swirled until the solution was clear.
5. Ethidium Bromide was added to the solution.
6. The gel electrophoresis box was assembled and the solution was poured in the box.
7. After the solution solidified, the “comb” was taken out and 1x TAE was poured over the gel until the gel was completely covered.
8. The gel mold was labeled, placed in a bag, and then put in the refrigerator.

Storage

After the DNA purification process was completed, the tubes were thrown away and the DNA  was placed in the freezer. The gel was placed in a bag and put in the refrigerator. The lab area was cleaned and additional equipment was  washed, cleaned, and placed back where it was found.

Conclusion/ Future Goals

At the end of lab, Mackenzie and I were able to make the gel mold, while Sydney  completed the DNA purification. In our next lab, w will run the DNA in the gel and see what our DNA length looks like after we stain the DNA and observe it with UV lights. We can now be sure that are DNA  samples are of good quality since most of the impurities have been eliminated. Also, I’m curious to see what our group’s DNA length looks like compared to the others.

Chandler Opara

2/7/19

Objective

The objectives of the lab were to identify our trees, categorize the three elements of the soil, and extract DNA from our soil samples.

Purpose

The purpose of this lab was to gather more information about the soil sample and the characteristics of the soil and tree. We also started the DNA extraction process to prepare for the PCR in the upcoming labs.

Procedure

Tree Metadata

1. Obtain the 24-well plate and transfer sample drops onto a slide. Use a compound microscope to observe any ciliates.
2. Obtain the falcon tube from the previous lab. Take a ruler and measure the components of sand, clay, silt composition in cm. Use the soil composition chart to calculate the percent composition and record it in the notebook.
3. Using the leaf diagrams, identify the leaf’s margin, venation, and shape/arrangement and use the microscope to get a better observation of the leaf. These characteristics will help identify the tree’s genus and species.

DNA Extraction

4. Take 1 gram of dry soil from the collected soil bag and add the sample to 1 gram of the silica beads.
5. Use a mortar and pestle to grind the content for 5 minutes.
6. Place 10mg of powdered activated charcoal and 2mL of DNA extraction buffer to the soil-glass mixture. Cut the tip of 1000mL micropipette to allow better mixing. Transfer the mixture into a centrifuge tube.
7. Place the tube in an incubator at 65 ℃ for 10 minutes. After it is finished, place the tube into a centrifuge at 12000g for 5 minutes.
8. Transfer the clear liquid supernatant out of the tube into a new tube.
9. Repeat the centrifuge process if there is any soil in the liquid supernatant. Initial the tube and place it in the rack for refrigeration.

Data

No ciliates were found in 24-well plate.

Tree Metadata

Type of tree: Southern Live Oak

Scientific Name: Quercus virginiana

 Leaf Identification

Soil Texture

Overall height: 2.5 cm

Clay: (.1/2.5) x100= 4%

Silt: (.4/2.5) x100= 16%

Sand: (2/2.5) x100= 80%

Soil Sample: Sand

Storage

The compound microscope was turned off, covered, and put away. Once the slides were cleaned, they were placed on a drying paper. The mortar and pestle were cleaned. The micropipettor tips were disposed in the designated cup. The falcon tube was stored in test tube rack at room temperature. Leaves were placed in bags and will be frozen for future use. The soil sample was stored in the class container under the fume hood.

Conclusion and Future Steps:

In conclusion, the information we gathered from classifying our tree and observing the leaf and soil will help us better understand the rhizosphere of the tree. We can also learn how the soil ciliate biodiversity is affected by these environmental factors. I was unable to see any new ciliates in the 24-well plate. I am glad that I was able to identify our tree with the help of Dr.Adair’s book and our leaves. We decided to use Sydney’s soil for the DNA extraction because her soil had a lot of ciliates and wanted to see how that can affect the DNA of the soil.