Sara Rothrock

4/25/2019

Purpose:

The purpose of this lab was to enter the metadata we collected this semester to determine which samples to sequence, to edit our poster presentations and write an abstract with our groups.

Procedure:

• Vote on a logo for stickers and t-shirts
• Enter the metadata we collected into the class excel sheet
• Revise our poster and write our abstract

Metadata:

Abstract:

There are many standard methods for extracting Ciliate eDNA but not all are effective and cost efficient.

Ciliates are protozoa in the Kingdom Protista. They play many different roles within soil and aquatic ecosystems. There has been very little research done on ciliate behavior and interactions within the soil. Our study targeted ciliates within the rhizosphere. The rhizosphere plays a key role in the cycling of nutrients as well as plant growth. Ciliates shape the diversity of microorganisms within the rhizosphere, thus their presence is vital to a healthy ecosystem.

The purpose of this study was to investigate new methods for eDNA extraction of ciliates.

Creating this new method was achieved by researching different ways to extract eDNA. These different methods were then combined to maximize the amount of eDNA collected. Metadata was collected regarding the soil sample and the environment it was collected from. Gels were run to determine the amount of DNA collected. The samples that contained an abundance of DNA underwent PCR in order to amplify the strand so they could more easily be sequenced.

The samples were loamy sand, with a pH of 6.5. The concentration of DNA in the sample was 1125 ng/ul and the A260/A280 value was 1.33. The DNA sequenced not only belonged to ciliate species, but fungal and bacterial species as well.

These results gave reason to believe the use of charcoal and Silica beads was effective in the extraction of DNA.

Poster:

Conclusion:

This lab was extremely beneficial because I learned how to properly edit my poster to have all the information necessary about the research without too many words. My group was marked down mainly because the conclusion had to have more detail but edit out information that can be conveyed during the presentation and our introduction has irrelevant information to the specific research we did. After revising our poster, I feel confident in the poster we submitted and we were able to fix all the mistakes we had made.

Future Steps:

In the future, I hope to continue doing research and that my metadata is selected to be further sequenced because I think that it. would be really cool to see what is in my specific sample.

Sara Rothrock

4/18/2019

Purpose:

The purpose of this lab was to analyze the soil eDNA we collected from our soil samples and extracted DNA from.

Procedure:

1. Before beginning eDNA analysis, ensure that Qiime2 is installed on the computer you will be using. If not downloaded, then download Qiime2 again.
2. After ensuring Qiime2 is downloaded, download the folder that contains all the data from the box link.
3. Activate Qiime2:
   • source activate Qiime2-2019.1
4. Switch the directory to the folder CILI-CURE_2018:
   • 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. Demultiplex the sequences:
   • 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 \

   • qiime demux summarize \–i-data demux.qza \

     –o-visualization demux.qzv

7. Denoise using DADA2 to 220 bases:
   • 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

8. Create a Feature Table:
   • 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

   • qiime metadata tabulate \

     –m-input-file denoising-stats.qza \

     –o-visualization denoising-stats.qzv
9. Create a phylogenic tree:
   • 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

10. Create a chart with Taxonomic classification:
    • 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

Results:

Demultiplexed-

Denoised-

Taxonomy Table-

Taxonomy Barplot-

Conclusion:

Overall, this lab was very successful because the only issues I had was having to re-download the Qiime2 program onto the computer. I feel much more confident using Qiime2 and using computer terminals to download files. Also, I tried the bonus to try and understand the programming better and learn about new codes for alpha and beta. In this lab, I also discovered that there was a ciliate that was recently discovered in Northern China that was in our soil sample. The ciliate did not have a food vacuole and it’s diet seemed to be of smaller microorganisms. I thought this was extremely fascinating and I am excited to further analyze our data and see what other types of ciliates there are in our soil sample.

Future Steps:

In the future, I would like to continue learning how to do metabarcoding and to expand my knowledge on data analysis.

Sara Rothrock

04/11/2019

Purpose:

To understand how use Qiime2 to analyze data sets which we will use in the future for our own data collected.

Procedure:

1. Open up the Terminal on computer
2. Check to ensure that miniconda and Qiime2 are downloaded on computer by typing in “source activate Qiime2-2019.1”. If the programs are not downloaded refer to last lab to re-download the programs.
3. After checking that Qiime2 is installed, type “conda activate qiime2-2019.1” to switch to the conda environment.
4. Then type “pwd” to ensure you are on the correct working directory.
5. Follow the instructions for the “Moving Picture” tutorial
   1. To create a new directory, enter:
      • mkdir qiime2-moving-pictures-tutorial
        cd qiime2-moving-pictures-tutorial
   2. To enter the data that you will analyze, enter:
      • wget \
        -O “sample-metadata.tsv” \
        “https://data.qiime2.org/2019.1/tutorials/moving-pictures/sample_metadata.tsv”
   3. Create another new directory by:
      • mkdir emp-single-end-sequences
   4. Download the two files:
      • wget \
        -O “emp-single-end-sequences/barcodes.fastq.gz” \
        “https://data.qiime2.org/2019.1/tutorials/moving-pictures/emp-single-end-sequences/barcodes.fastq.gz”
      • wget \
        -O “emp-single-end-sequences/sequences.fastq.gz” \
        “https://data.qiime2.org/2019.1/tutorials/moving-pictures/emp-single-end-sequences/sequences.fastq.gz”
   5. Make the files into an artifact:
      • qiime tools import \
        –type EMPSingleEndSequences \
        –input-path emp-single-end-sequences \
        –output-path emp-single-end-sequences.qza
   6. Demultiplex the sequences using:
      • qiime demux emp-single \
        –i-seqs emp-single-end-sequences.qza \
        –m-barcodes-file sample-metadata.tsv \
        –m-barcodes-column BarcodeSequence \
        –o-per-sample-sequences demux.qza
   7. Generate a visualization of the data by making the files into a qzv:
      • qiime demux summarize \
        –i-data demux.qza \
        –o-visualization demux.qzv
   8. Drag and drop the qzv file from Finder into Qiime2 View to create a visual interactive quality plot.
   9. To ensure the quality of the data, we will cut off the data around 120 by denoting the data:
      • qiime dada2 denoise-single \
        –i-demultiplexed-seqs demux.qza \
        –p-trim-left 0 \
        –p-trunc-len 120 \
        –o-representative-sequences rep-seqs-dada2.qza \
        –o-table table-dada2.qza \
        –o-denoising-stats stats-dada2.qza
      • qiime metadata tabulate \
        –m-input-file stats-dada2.qza \
        –o-visualization stats-dada2.qzv
      • mv rep-seqs-dada2.qza rep-seqs.qza
        mv table-dada2.qza table.qza
   10. Drag and drop the stats-dada2.qzv file from Finder into Qiime2 View to observe the data.
   11. Then, perform taxonomic analysis on the data:
       • wget \
         -O “gg-13-8-99-515-806-nb-classifier.qza” \
         “https://data.qiime2.org/2019.1/common/gg-13-8-99-515-806-nb-classifier.qza”
   12. Next, run these commands to convert the taxonomy file to a qzv:
       • qiime feature-classifier classify-sklearn \
         –i-classifier gg-13-8-99-515-806-nb-classifier.qza \
         –i-reads rep-seqs.qza \
         –o-classification taxonomy.qza
       • qiime metadata tabulate \
         –m-input-file taxonomy.qza \
         –o-visualization taxonomy.qzv
   13. Drag and drop the taxonomy qzv file from Finder into Qiime2 View to analyze the confidence level of samples.
   14. To create a taxa bar plot use:
       • qiime taxa barplot \  –i-table table.qza \  –i-taxonomy taxonomy.qza \  –m-metadata-file sample-metadata.tsv \  –o-visualization taxa-bar-plots.qzv
   15. Drag and drop the bar plot qzv file from Finder into Qiime2 View to observe the taxa levels.
   16. Save all files from the qiime2-moving-pictures-tutorial

Conclusion:

This lab helped us learn and become more comfortable using a computer terminal by working through the Qiime2 moving picture tutorial. This taught us how to create different graphs and bar plots that can be used to visualize our data. Also, after this lab I am more comfortable using computer terminals and using various commands. In the future, I hope to continue learning about bioinformatics and coding on computers because I believe bioinformatics will become more common with advancements in medicine.

Sara Rothrock

3/29/2019

Purpose:

The purpose of this lab was to be introduced into Illumina sequencing to be prepared for future data analysis of our results.

Procedure:

• Organize the cards in order to complete the QTM

Sequencing Order:

1. Break up genomic DNA into more manageable fragments of around 200 to 600 base pairs by using enzymes or by PCR.
2. Tag the DNA fragments with short sequences of DNA called adaptors.
3. Denature the double-stranded molecules into single-stranded molecules that have the adaptor and primers binding site. This is done by incubating the fragments with sodium hydroxide.
4. Attach the DNA fragments to the flow cell through complementary binding of the adaptors to the oligos (primers) on the surface of the flow cell.
5. Replicate the DNA attached to the lyocell to form small clusters of DNA with the same sequence.
6. Unlabeled nucleotide bases and DNA polymerase are then added to lengthen and join the strands of DNA attached to the flow cell. This creates ‘bridges’ of double-stranded DNA between the primers on the flow cell surface.
7. The double-stranded DNA is then broken down into single-stranded DNA using heat, leaving several million dense clusters of identical DNA sequences.
8. Primers and fluorescently labeled terminators (a version of nucleotide base- A, C, G, or T- that stop DNA synthesis) are added to the flow cell.
9. The primer attaches to the DNA being sequenced.
10. The DNA polymerase then binds to the primer and adds the first fluorescently-labeled terminator to the new DNA strand. Once a base has been added no more bases can be added to the strand of DNA until the terminator base is cut from the DNA.
11. Lasers are passed over the flow cell to activate the fluorescent label on the nucleotide base. This fluorescence is detected by a camera and recorded on a computer. Each of the terminator bases (A, C, G, and T) give off a different color.
12. The fluorescently-labeled terminator group is removed from the first base and the next fluorescently-labeled terminator base can be added alongside. And so, the process continues until millions of clusters have been sequenced.

Conclusion:

In this lab, I learned the proper order of Illumina sequencing and understood why each step happens by discussing with my partner and Dr. Adair. The game helped me critically think about each step and why it happens. I feel prepared to move forward in our data analysis of our results in the future.

Sara Rothrock

3/8/2019

Purpose:

The purpose of this lab was to run our samples through gel electrophoresis to ensure our PCR samples meet the qualifications to be sent off and sequenced.

Procedure:

• Obtain a 1.5% agarose gel prepared from the previous lab
• Place the gel into the gel electrophoresis box (wear gloves)
• In the first well micropipette 5 μl of 1 kb ladder
• In the second well micropipette 10 μl of your DNA treatment sample
• In the third well micropipette 10 μl of your DNA control sample
• Then after each well is loaded, write down what is in each well and run the gel electrophoresis for 30 minutes at 100V
• After the gel finished, Dr. Adair analyzed the samples with MCB C305

Results:

Conclusion:

From the gel electrophoresis, our group was able to determine that our DNA sample has a large amount of DNA. Now we need to ensure our DNA is up to standards in order to have our DNA sample sequenced. If we are able to get our DNA sequenced, we will be able to have a deep understanding of the types of ciliates there are in our soil and be able to relate it to the overall health of our trees at Baylor.

Future Goals:

In the future, I hope to continue practicing and improving my various lab skills. Also, I hope to learn more about various diseases that could kill trees that are seen on Baylor’s campus.

Sara Rothrock

2/28/2019

Purpose:

The purpose of this lab was to prepare our DNA samples to be amplified through PCR and to begin collaborating on our poster design.

Procedure:

1. Using the information from the Nanodrop last lab, calculate the amount of DNA needed to get 100 ng. Have TA or Dr. Adair check your calculations before moving on.
2. Obtain two PCR tubes with 12.5  μl of 2x Master mix already in them
3. Add 1 μl of 10 μM primers to each PCR tube
4. Label one of the PCR tubes control and experimental
5. In the experimental tube, add your calculated amount of DNA to the tube and add as much water needed to get to 25 μl total in the tube
6. In the control tube, add 11.5 μl of water and do not add any DNA to this tube
7. Centrifuge both tubes for 10 seconds to make sure all the contents are mixed at the bottom of the tube
8. Then, store both tubes on rack to be put into the thermocycler

Results:

PCR Tube Volumes:

Conclusion:

This lab we learned how to prepare our DNA samples for amplification through PCR and practiced our calculations used to create different concentrations and dilutions. Then, we review on Kahoot and in each group we discussed our plan for our poster project. For my group, we did not come across an issues or errors in the preparation of our samples.

Future Goals:

In the future, I hope to learn about sequencing DNA from different organisms and learn more about how ciliate DNA can compare to different sequences in the genome database.

Sara Rothrock

2/21/2019

Purpose:

The purpose of this lab was to run our extracted DNA through gel electrophoresis to determine the concentration of DNA in the sample. We also used the nano drop and an imaging software to analyze the extracted DNA.

Procedure:

Gel Electrophoresis-

• Take one gel that was made last lab and remove the rubber stoppers from the side
• Place the gel inside the gel electrophoresis box (be sure the wells are on the negative side of box) and fill with TAE buffer so the gel is fully submerged
• Take the purified DNA from the previous lab and put 9 μl of the purified DNA into a new centrifuge tube
• Add 1 μl of 10x loading buffer to the purified DNA in the centrifuge tube
• Place the centrifuge tube into a microcentrifuge for five seconds to mix the DNA and loading buffer
• Micropipette out the 10 μl and place into one of the wells of the gel
• Micropipette 5 μl of a small mass standard into another well
• Micropipette 5 μl of a large mass standard into another well
• Then run the gel at 100 volts for 20 to 30 minutes
• Afterwards, take the gel to the UV illuminator to observe the concentration in your sample

Nanodrop-

• Clean the machines and place 1 μl of your purified DNA on top of holder
• Then let the nano drop measure the concentration of DNA and record results

Results:

Gel Electrophoresis-

NanoDrop-

Conclusion:

This lab was successful because our group found a large amount of DNA in our sample. However, our DNA is not very pure because our numbers were close to 1.3- 1.4 when they should have been close to 1.8. I was worried that it would not work at first because we had an air bubble in one of the wells but overall our trial turned out to be successful.

Future Goals:

In the future, I would like to learn more ways to purify DNA so if I purify DNA again the A260/A280 number will be closer to 1.8 in a nano drop machine.

Sara Rothrock

2/14/2019

Purpose:

The purpose of this lab was to purify our DNA samples and to create agarose gels to run gel electrophoresis trials on our DNA samples.

Procedure:

DNA Purification-

• Take 1 mL of crude DNA sample and add to a 15 mL tube
• Add 2 mL of DNA Resin that is at 37°C to the 15 mL tube
• Setup a syringe column and barrel
• Add the DNA and Resin to the column inside the vacuum hood
• Take 1500 μl of the solution and add 2000 μl of isopropylene to clean the solution then add the rest of the solution
• Transfer sample into a 1.5 mL tube and place in centrifuge for 5 minutes at 8000 g
• Take column out of the tube and put in 80°C heat block for about a minute
• Transfer to another 1.5 mL tube and add 50 μl of sterile H2O heated to 80°C
• Incubate the tube for one minute and spin at 800 g for one minute
• Label with group names and section then store

Gel Electrophoresis-

• Add 4 mL of 10X TAE into a conical vile and add 36 mL of DI water into the conical vile
• Measure out 0.4 g of agarose powder and place into an Erlenmeyer flask
• Add the 10xX TAE and DI water solution into the Erlenmeyer flask
• Microwave the solution for one minute
• After microwaving, swirl the solution for about a minute (be sure to wear gloves while swirling, glass is hot)
• Add 2 μl of Et Br to solution and swirl once
• Pour the solution into a gel electrophoresis box and allow to cool for 30 minutes
• Label the box with your group members and section on the side of the box using tape
• After the gel has solidified cover the top of the gel with 1X TAE so the gel will not dry out during storage
• Place in ziplock bag and store in a refrigerator

Storage:

The gels and DNA samples were placed into a refrigerator to keep them cool.

Conclusion:

This week in lab we purified our DNA samples and created our agarose gels for gel electrophoresis tests. My group was able to successful complete both tasks and we are ready for our DNA testing next lab.

Future Goals:

In the future, I hope to practice more DNA extraction methods and to learn more about how to extract DNA from encysted ciliates from soil samples.

Sara Rothrock

2/7/2019

Purpose:

The purpose of this lab was to calculate the soil texture of our sample, identify the species of our tree and extract DNA from the ciliates we cultured in the previous lab. Also, in this lab it was important to consider potential the workflow of our research.

Procedure:

Determining Soil Texture:

• Take the soil sample in the test tube from the previous lab and measure each layer in cm (sand is on the bottom, clay is in the middle and silt is on the top)
• After you have measured each layer then, calculate the percent composition of the sand, clay and silt
  • Divide the mass of each layer by the total mass of the soil and multiply by 100
• Use the soil texture triangle diagram to determine soil texture

DNA Extraction:

• Take 1 gram of dry soil and 1 gram of glass beads and grind in a mortar and pestle for 5 minutes
• After the mixture is grinned finely, add 2 mL of DNA extraction buffer and a small amount of activated charcoal powder
• Then stir the mixture until it is evenly mixed and transfer into a 2 mL eppendorf tube
• Incubate the tube at 65°C for 10 minutes
• Place in centrifuge at 12000g for 5 minutes (Be sure the centrifuge is balanced before turning on)
• Transfer the supernatant into a separate eppendorf tube and refrigerate

Results:

Soil Texture-

The soil texture was sandy clay

Conclusion:

In this lab, we checked the ciliates we attempted to culture last week and were successful in a few of our cultures. However, the ciliates are very small and were difficult to find. Also, I determined my soil texture was sandy clay and the species of my tree was a Live Oak or Quercus virginiana. My group’s tree is a live oak because we compared our leaf structures to the Texas A&M Forest Service log of Trees in Texas and our leaf’s characteristics were similar to that of the live oaks.

Future Goals:

In the future, I hope to continue practicing and improving various lab techniques and learn more about DNA extraction techniques that can be used in the future. Also, I would like to learn how or find a method to extract DNA from ciliates that have encysted in the sample.

Sara Rothrock

1/31/2019

Purpose:

The purpose of this lab was to calculate the percent water content of and to find ciliates in the previously collected soil samples from last week.

Procedure:

Percent Water Content-

1. Weigh the “wet” soil Petri dish from the previous lab and record the mass
2. Then using the data collected from the previous lab, calculate the percent water content
   • Use this equation to calculate: (Wet Soil Mass – Dry Soil Mass)/ Wet Soil Mass x 100

Ciliate Discovery-

1. Take your non-flooded plate and observe under a dissecting microscope to search for ciliates
2. When you find a ciliate, grab a clean slide and micropipette 5 μl of the ciliates onto the slide
3. Observe under a compound light microscope and record the characteristics of the ciliates you find. Also try to photograph the ciliates found
4. After you have found some ciliates, take a 24 well plate and micropipette the ciliates from the slide into a well to begin a culture of the ciliates
5. Be sure to initial the cover of the well plate above the well you started your culture in
6. If you are unable to find any ciliates in your sample, ask a TA or Dr. Adair to help you find some ciliates in your sample.

Results:

Percent Water Content-

Ciliate Discovery-

I found a lot of different ciliates, most of them were very small and moved to quickly for me to photograph. Two ciliates that were notable was a large blue ciliate and a group of rectangular ciliates.

Conclusion:

This lab was very successful because my group was able to fill a 24 well plate to start ciliate cultures. Also, in my soil sample there was an abundance of ciliates and there was a lot of diversity amongst the ciliates. I started fourteen cultures in my group’s well plate and I am hopeful moving forward with all the ciliates I found in the lab.

Future Goals:

In future labs, I hope to learn more about DNA and RNA extraction methods and how to identify ciliates in different methods other than morphology.