Objective

The objective for this lab was to refine our posters and prepare our Abstracts so that we can present our findings next Friday. We also voted on a logo and ensured our metadata was entered correctly.

Purpose

The purpose of this lab was to prepare our presentations for the CILI-CURE Symposium next Friday. We also wrote a short Abstract that compressed the entirety of this semester’s research into a paragraph. Additionally, we checked our metadata to ensure the accuracy of our findings and hopefully have our sample be sent off for genetic sequencing.

Procedure/Data

When we first got to the lab, we voted on a logo for CILI-CURE. After this, we worked in our groups to compose an Abstract for our poster that was approximately 250 words long and that accurately depicted the research we had done in the last semester. Once this Abstract was completed, we focused on editing our poster and addressing comments that could be easily fixed. We altered the organization to make it more aesthetically pleasing and clarified the purpose of our research in the introduction and conclusion.

Conclusion

We were able to draft an accurate Abstract as well as edit the poster to be much better than before. I must say I am looking forward to presenting during the symposium, as I am confident in my knowledge of all the processes and procedures that we performed this semester.

Objective

The objective for this lab was to apply the skills we learned in the previous labs to analyze a data set.

Purpose

The purpose for this lab was to exercise our knowledge about Qiime2 and analyze a set of data in preparation for the analysis of our own.

Procedure/Data

As always, we bagan by installing Miniconda and Qiime2 on our computers. Then, we downloaded the folder containing all the data we would need. After changing the directory to that folder and activating Qiime2, we proceeded to import the sequences as a Qiime2 artifact using the code

qiime tools import \

–type EMPPairedEndSequences \

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

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

Once this was accomplished, we demultiplexed the sequences using the follwing codes.

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

We then used DADA2 to denoise the data and ensure our results are reliable.

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

After this, we created a feature table of our data and converted it into a .qzv file so it could be viewed.

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

Then, we created a phylogenetic tree that connected our DNA sequences to their respective classifications.

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

Finally, we created a taxa bar plot to display all this data in an interactive way that could better show all the specifics of the data.

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

Conclusion

Overall, this lab was much like last lab where we practiced using the Moving Pictures tutorial on the Qiime2 website. Practicing with Qiime2 is much needed, and I feel as though I am getting more comfortable using it. Additionally, my understanding of each step and why it is important is growing. I am excited to perform these anaylses on our own data in the future.

Objective

The objective for this lab was to learn more about the operation of Qiime by completing the “Moving Pictures” tutorial on their website. This takes us through each step in the process of generating an effective chart with all of the sequence data, metadata, and phylogeny.

Purpose

The purpose of this lab was to familiarize ourselves with the various commands and procedures that go into utilizing Qiime. We also learned new terminology and skills to aid in our understanding of the process.

Procedure/Data

Before beginning with the tutorial, we had to reinstall Qiime natively on our computers using the same process we learned last week. First, we downloaded the Miniconda 64-bit.pkg for the Mac OS. Then we opened a new terminal and input the codes “conda update conda” and “conda install wget” to ensure we are running the latest version of conda. We then installed Qiime withing the conda environment using the code “wget https://data.qiime2.org/distro/core/qiime2-2019.1-py36-osx-conda.yml
conda env create -n qiime2-2019.1 –file qiime2-2019.1-py36-osx-conda.yml” After this, we activate the conda environment with “source activate qiime2-2019.1” Once this was done, we moved on to the tutorial.

Obtaining the Data

• mkdir qiime2-moving-pictures-tutorial
  cd qiime2-moving-pictures-tutorial

This code makes a new folder named “qiime2-moving-pictures-tutorial” and opens it in the terminal so we may operate within it.

• wget \
  -O “sample-metadata.tsv” \
  “https://data.qiime2.org/2019.1/tutorials/moving-pictures/sample_metadata.tsv”

With this code, we can download (wget) the sample metadata that will be used in this tutorial.

• mkdir emp-single-end-sequences

This code will make a new folder in which we can put the single end sequences.

• 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”

Inputting this will download the single end data from the website into the new folder we made.

• qiime tools import \
  –type EMPSingleEndSequences \
  –input-path emp-single-end-sequences \
  –output-path emp-single-end-sequences.qza

This code will import the sequence data into a Qiime artifact (.qza).

Demultiplexing Sequences

• 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

The demultiplexing sorts the data into individual samples. This associates each barcode sequence with each sample.

• qiime demux summarize \
  –i-data demux.qza \
  –o-visualization demux.qzv

This will generate a summary of the demulitplexing results that we can visualize and makes sense of ourselves.

Denoise

For this tutorial, we used the DADA2 method for implementing quality control in our sample.

• 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

Running this code will cut off the lowest quality sequences to ensure our data is accurate and reliable. It generates a .qza file as a result.

• qiime metadata tabulate \
  –m-input-file stats-dada2.qza \
  –o-visualization stats-dada2.qzv

This code will convert the .qza file into a more useful .qzv file so we can view the results.

• mv rep-seqs-dada2.qza rep-seqs.qza
  mv table-dada2.qza table.qza

This code renames the files so they can be found and identified more easily.

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

Running this code will take our demultiplexed data and make a feature table out of it. It will also immediatley convert it to a .qzv file so we can view it.

Taxonomic Analysis

• 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”

With this code, we can download the taxonomic data necessary to classify organisms based on our sample DNA.

• 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

These two codes, when entered together, will integrate our data with the taxonomic data to associate the matching barcodes. Since it also generates a .qzv file, we can also put this in the Qiime viewing tool to visualize it.

• qiime taxa barplot \
  –i-table table.qza \
  –i-taxonomy taxonomy.qza \
  –m-metadata-file sample-metadata.tsv \
  –o-visualization taxa-bar-plots.qzv

With this final code, we compile all of our data and metadata to create an interactive bar plot that displays our findings in countless different forms, with many different filters and organization styles.

Conclusion

Although it may have been somewhat difficult to perform, this procedure turned out to be very interesting and useful to our research. The final product was well worth the work we had to go through, putting in several codes and dealing with the occasional error message. I found myself enjoying the process after a while, seeing the results slowly build until the final culmination of the bar plot. I feel as though I learned a lot during this week’s lab and I am eager to see how we can utilize these skills in our own work.

Objective

The objective for this lab was to learn about the software used to analyze our sequenced data. We would then install this software on our computers for later use.

Purpose

The purpose of this lab was to introduce us to the skills we will need to analyze our data and ensure we have the proper software to do so.

Procedure/Data

Terminal:

There is a function on Mac computers called Terminal in which one can input commands to navigate the files on the computer and perform simple tasks. These commands range from “cd ~/Documents” to open up the Documents folder to ” man [command]” to learn more about a specific command.

Installing Qiime 2:

To install Qiime 2, you must first download an environment for it to run in. The environment we used was Miniconda. After installing miniconda, ensure you have the latest version by inputting the “wget” command in the Terminal to download it. Now, within Miniconda, download the Qiime 2 for the Mac OS/OS X (64 bit) version. After we had installed Qiime 2, we can input the ” –help” code to see a list of commands for this program. Then we were given a brief example of what a finished project will look like.

Conclusion

In conclusion, this lab was admittedly pretty confusing. I am not very proficient in the use of computers, and coding has always seemed to be too lofty a goal for me. I do feel as though I have learned enough to continue further with the project. I am also eager to see what Qiime 2 does with our data and what we will be able to discern from it.

Objective

The objecive for this lab was to present the rough draft of our poster that we had constructed during the latter portion of the last lab.

Purpose

The purpose of this lab was to receive feedback on the rough draft so we can correct any wrongs and ensure we have the correct information needed on it. We also got to talk to our groupmates and discuss how we were going to fix it and formulate a plan to finish it.

Data

Conclusion

Our poster needs much work. In short, we need to change the title, insert the second gel electrophoresis picture, label both of those figures, take pictures of our tree and the book, make a flow chart for the methods, and find sources to reference.

Objective

The objective for this lab was to run our DNA sample along with the control against the DNA ladder to determine the size of our DNA molecules. Then, we went up to the computer lab to take the midterm quiz and start working on our scientific posters.

Purpose

The purpose of this lab was to acquire more data about our DNA sample to see if it was worth sending off to be sequenced. We also took our midterm quiz, and after we worked on our posters that we will craft over the next few weeks.

Procedure

1. Set up the 1.5% agarose gel
2. Run the gel electrophoresis at 100v for approximately 30 minutes.
3. Go to computer lab for midterm quiz
4. Work on poster and QTM

Data

Conclusion

Our sample, found in lane 4, showed that we do have DNA that has the right section which we will analyze, meaning that it is a viable candidate for PCR. In future labs, we will work as a group to prepare a professional poster to report our findings.

Objective

The objective for today’s lab was to calculate exactly how much water and purified DNA we would need to add to our solution in order to run a polymerase chain reaction. We also reviewed for our midterm and began organizing our posters.

Purpose

The purpose of the PCR was to amplify our DNA sample so we would have a sufficient amount of DNA to send off for sequencing. We were also able to review for our midterm as a class through Kahoot, and we could ask any clarifying questions that we needed answered. Finally, we were able to work with our group to design a poster that we will create using all the data that would go in our final scientific paper.

Procedure

First, we had to do the calculations. For both the sample and control we needed a final volume of 25 µL, 1µL of 10µM primers, and 12.5 µL of 2x taq mix. The control, however, contained no DNA and 11.5 µL of water, while the sample contained 1.52µL of DNA and 9.98µL of water. These two solutions were made and placed in a rack with the rest of the students’ to undergo PCR. After this we did our QTM’s and reviewed using Kahoot, in which I almost got first (I was in the lead until the 9th question). Then we focused on organizing and titling our poster, which was simple enough in itself.

Data

After our calculations, we found that, to obtain a final volume of 25µL in the sample solution, we needed to add 1.52µL of DNA and 9.98µL of water. Other than that, there was not much data to obtain from this lab. The useful data will come in when the DNA is sequenced and we can analyze it.

Conclusion

This lab was mainly a simple preparation of our DNA for the polymerase chain reaction procedure. In addition to this, we did get to review for the midterm. I believe this was very much needed as many of us did not know how to prepare for the exam. Finally, we got to work with our groups to begin the poster that we will have to construct, which proved relatively simple since my group was in agreement about all the aspects we discussed.

Storage

Our solutions were labelled and placed with everyone else’s at the front of the classroom. Near the end of lab, Dr. Adair took these upstairs to beging the PCR process. Our work space was sanitized with bleach before we began making our solutions and was wiped down thoroughly. As per usual, I was the last person in my group to exit lab, so I made sure that our area was neat and everything was put away as it should be.

Objective

The objective for this lab was to perform gel electrophoresis using the gel’s we created and the DNA sample that was purified the previous week. Once the gel electrophoresis was performed, we could interpret the results. Also, we performed a nanodrop experiment, which showed how much light was absorbed by the DNA in the droplet of sample.

Purpose

The purpose of performing the gel electrophoresis was to separate the DNA of the sample according to its molecular size. By doing this and comparing it to the mass standards, we were able to determine the approximate quantity of DNA that was present in our sample. Additionally, the nanodrop served to validate our findings and give us a similar estimate of the concentration of DNA in our sample.

Procedure

Gel Electrophoresis

Firstly, we placed the gel in the buffer chamber, ensuring it was oriented correctly and covered with 1x TAE buffer. Next, we placed 9 microliters of our DNA sample into a small microfuge tube mixed it with 1 microliter of 10x loading buffer for a total of 10 microliters of 1x buffer/DNA solution. After practicing loading gels for a while, we took 10 microliters of the solution in the microfuge tube and carefully pipetted it into well #2 of the agarose gel. Then we placed 5 microliters of the low (31) mass standard and 5 microliters of the high (500) mass standard into wells #4 and #5, respectively. Lastly, we placed 10 microliters of group 3’s sample in well #7 and ran the gel electrophoresis for 20 minutes at 100 volts.

DNA Analysis

Once the gel electrophoresis was finished, we brought our gel up to the 3rd floor so it could be analyzed under an ultraviolet light. Under this light, the DNA strands could be seen and compared to the mass standards to determine an approximate mass of DNA in our sample. We also ran a nanodrop test by placing an extremely small drop of sample onto the anakyzer. This is what determined the concentration of DNA by analyzing how much light was absorbed by the sample.

Data

Our DNA, or group 1’s DNA, was found to more resemle the low mass standard of 31, while group 3’s sample looked much more like the high mass standard of 500. Through the nanodrop procedure, our sample’s density of DNA was found to be approximately 65.8 nanograms per microliter, and it had an A260/A280 of 1.46 and an A260/A230 of .32.

Conclusion

Our DNA sample did not contain as much DNA as some others, but we did still confirm the presence of DNA in our sample. This is a resounding success, as those who did not find any DNA must repeat the procedures of the last 3 labs. Since we have DNA in our sample, we will put our sample through PCR to multiply the present DNA. This is so that we may analyze the DNA and sequence it without worrying about damaging or using up all of the sample DNA.

Storage

At the end of lab, we cleaned the containers that held the gel with bleach, and we gave our DNA sample and our gel to the instructors for storage. I ensured my station was clean and nothing on our table was left plugged in.

Objective

The objective of this lab was to purify our eDNA sample that we extracted the previous week. In addition to removing all of the debris from the eDNA sample, we also made an agarose gel that we can use for gel electrophoresis next week.

Purpose

The purpose of purifying the eDNA was to remove any microscopic debris that we extracted along with the genetic material. The debris could cause issues with the gel electrophoresis, so we needed a pure eDNA sample. The purpose of making the gel was simply that it is the medium we will require to run a gel electrophoresis next week.

Procedure

DNA Purification

• Add deionized (DI) water to the crude soil DNA extract up to the 1mL mark. Put this 1mL of solution into a clean 15mL tube appropriate for mixing.
• Add 2mL of warm (37 degrees Celsius) DNA resin from the Wizard PCR Prep DNA Purification kit.
• Set up a column and syringe barrel on a vaccuum filtration manifold.
• Add half of the mixture in your 15mL tube to the barrel. Once all the liquid has been pulleed through, add the other half.
• Wash the column by adding 2mL of 80% isopropanol to the barrel. Add 1mL at a time up to 6mL total (3 total washes, 2 mL per wash)
• Remove the column from the syringe barrel and place in a clean 1.5 mL centrifuge tube. Spin at 8000 x g (or 8000 rcf) for 5 minutes.
• Take the column out of the centrifuge tube, disopse of the tube, and place the column in a new 1.5 mL tube, heating it for 30-60 seconds in an 80 degrees Celsius heat block.
• Add 50 microliters of DI water to the column and let it incubate for 1 minute.
• After the minute is up, spin again at 8000 x g for 1 minute.
• Take the tube out, and dispose of the column.
• Label the tube and place it in the freezer for storage.

Data

No data was recorded in this lab; it was mostly just setting the stage for the gel electrophoresis we will run next week.

Conclusion

This lab was definitely one of the longest. We all had to figure out the procedure and wait for our processes to be done, but it was worth all the work in the end. After all, purifying the DNA is an essential step. While my partner Anthony and I were purifying our eDNA, my other partner Emely was preparing the agarose gel we are going to use next week.

Storage

Once purified, our DNA samples were put in the freezer to prevent them from denaturing. I ensured that our station was neat and all of our trash was disposed of accordingly.

Objective

The objective for this lab was to extract DNA from our soil samples. Additionally, we calculated the percent composition of the soil in terms of sand, silt, and clay. Also, we analyzed leaves we gathered from our tree to define its genus and species.

Purpose

The purpose for extracting DNA is so we can arrange it to observe how many different species of microorganisms are in that specific soil environment. We calculated the compostion of the soil in order to be able to associate any ciliates we find with that type of soil. We identified the type of tree we got the soil from for this same purpose.

Procedure

At the beginning of lab, we took a few minutes to look in our cultures for ciliates. After that, we carefully observed our leaves and all the different characteristics of them, such as venation, margins, and overall shape. Using guides online and a book, we identified the type of tree from which these leaves originated. Our soil composition test followed. We took our falcon tubes full of the vortexed soil from las week and measured in millimeters the layers of sand, silt , and clay. We then moved on to the actual DNA extraction from our soil samples, where we first measured one gram of soil and one gram of silica beads and ground them in a mortar and pestle until they were a fine powder. We added 2 milliliters of DNA extraction buffer and approximately 10 milligrams of activated charcoal to the mortar and pestle, where we mixed it using a pipette tip. Once we were done mixing, we transferred the contents to a centrifuge tube. The tube was incubated at 65 degrees Celsius for 10 minutes then centrifuged at 12000g for 5 minutes. We transferred the supernatant liquid into a new centrifuge tube then centrifuged that tube as well to be sure that we had no dirt particles in our sample of DNA.

Data

My soil was found to be approximately 68.75% sand, 12.5% silt, and 18.75% clay. This can be classified as sandy loam. I classified my tree as a live oak, or Quercus virginianus. We successfully prepared a sample of DNA using Emely Canas’ sample of soil.

Conclusion

Looking into the cultures I made, I could only see small organisms and not much of what I thought to be a ciliate. Identifying my tree was a little complicated, as not all of my leaves looked the same despite them all being from the same branch. My soil composition was easy enough, as my tube showed clear lines to distinguish between the sand, silt, and clay. The DNA extraction was the longest yet most rewarding part. I am excited to analyze our samples and see how we can use metabarcoding ourselves.

Storage

I was one of the last students to leave the lab because I was finishing the Questions That Matter, so my lab partners did most if the cleaning and storing for me. I did check to make sure the centrifuge was empty and I looked over the microscopes to check that they were all unplugged and covered. My leaves were stored with the rest of the class’s for later use and the mortar and pestle we used were washed in the sink and left our to dry.