Purpose: The purpose of this lab was to discuss how we improve and revise our posters using the comments we received from the TA/instructor, and input our soil meta-data into a shared excel sheet. We also gathered all the information and samples we had so we can send our samples to be sequenced so it can analyzed later.

Procedure:

1. Vote on Ciliate logo
2. Fill out soil meta-data excel sheet
3. Do final edits on poster for presentation next week
4. Revise and edit abstract for the poster
5. Insert poster and abstract into class Box file

Soil Meta-data:

Meta-data 1:

Meta-data 2:

Poster:

Abstract:

Title: Analysis of eDNA from a Soil Sample in the Search for Soil Ciliate DNA

Soil ciliates are crucial organisms in the rhizosphere as they perform many important roles contributing to soil health. Currently, research concerning topics related to soil ciliate diversity is limited. Conventionally, ciliates have been identified and categorized through morphological characteristics and behaviors due to a lack of a standard genomic method for easy identification. This study aims to explore a standard method for ciliate identification through metabarcoding. For this, soil was collected and environmental DNA was extracted using the Silica Bead grinding method followed by Column Purification. Polymerase Chain Reaction was used to amplify the V4 region of the 18s rRNA subunit. Gel electrophoresis results showed that PCR amplified the eDNA. The image taken of the gel showed a concentrated band of DNA from the eDNA sample. Nano-drop analysis of the eDNA showed a 250.2 ng/ml of the DNA while it had a purification of 1.46 A260/280. The eDNA extraction method conducted appears to be the best option for extracting DNA due to the amount of DNA replicated. Metabarcoding of the V4 region of the 18s rRNA subunit was conducted using the eDNA to identify what kind of ciliates were extracted from the environmental sample and sequence. Bioinformatics (Cyverse/QIIME2) will be used to analyze how successful the V4 region can be in the identification of ciliates.

Conclusion/Future Steps: After this lab, we plan to do a practice presentation of our poster to the class next week to do last minute critique for how its presented. We also plan on presenting the poster at CURE so we can share our data to other people on what we learned from our research. After we send our samples to be sequenced, we can later analyze these samples using Cy-verse/QIIME2, seeing what kind of ciliates were found in the DNA solution. This lab should prepare us for the presentation so we know all the information we must present to our peers. This lab help us make crucial changes to the poster so we won’t make any major mistakes on our poster presentation.

Purpose/Objective: The purpose of this lab was to be able to run metabarcoding analysis using Qiime 2 on the Apple computer (Terminal) to analyze the data set obtained from the eDNA soil sample. Using this data analysis, we’ll be able to determine what kind of organisms we have in our samples and if their any ciliates sequenced in the sample. We’ll be analyzing 100,000 reads.

Procedure:

Reinstall Qiime:

1. Download miniconda from miniconda website (64 bit .pkg)
2. input conda updata conda into terminal
3. input conda install wget
4. input 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
5. input source activate qiime2-2019.1

QIIME Walkthrough:

1. Open up the terminal
2. change the directory to the fold your using. Input cd CILICURE-2018 mkdir
3. activate QIIME
4. Importing sequences as QIIME2 artifact:
   1. Input: qiime tools import/, type EMPPairedEndSequences/, input-path emp-paried-end-sequences/, output=path emp-paired-end-sequences.qza
5. Demultiplexing sequences
   1. 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
6. Denoising using DADA2 and creating a feature table with Representative sequences
   1. qiiime 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
   2. qiime feature-table summarize/, I-table table.qza/, o-visualization table.qzv/, m-sample-metadata-file sample-metadata.tsv
   3. qiime feature-table tabulate-seqs/, I-data rep-seqs.qza/, o-visualization rep-seqs.qzv
   4. qiime metadata tabulate/, m-input-file denoising-stats.qza/, o-visualization denoising-stats.qsv
7. Creating a phylogentic tree
   1. 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
8. Taxonomic Classsficaiton
   1. qiime feature-classifier classify-skleam–I-classifier silva-132-99-515-806-nb-classifier, reads rep-seqs.qza–o-classification taxonomy.qza
   2. qiime metadata tabulate/, m-input-file taxonomy.qza/, o-visualization taxonomy.qsv
   3. qiime taxa barplot/, I-table table.qza/, I-taxonomy taxonomy.qza/, m-metadata-file sample-metadata.tsv/, o-visualization taxa-basr-plots.qzv

Results:

Conclusion: After this lab, we were able to create a phylogenic tree and see what kind of ciliates or other organisms were found in the samples of eDNA or Chelex samples in the 100,000 data set. Using Qiime 2 view allowed us to view the data from any .qzv file that we created using QIIME. We were able to see that there were ciliates in the sample and we decided to pick one of them and look into what kind of characteristics is unique to that ciliate. In the future, we will be using ATMOSPHERE to analyze large data sets and analyze them using data analysis of sequences

Purpose: The purpose of this lab was to learn more commands in Qiime and in the terminal app. We used the “Moving Picture” tutorial commands to make new folders, make new files in those folders, and viewing those files using the Qiime 2 view website to view our data.

Procedure:

1. Install Miniconda and Qiime using the instructions from the Qiime website
2. Follow the instructions for “Moving Picture” tutorial
   1. Create a new directory and change to it:
      1. mkdir qiime2-moving-pictures-tutorial
         cd qiime2-moving-pictures-tutorial
   2. Explore sample metadata
      1. wget \
         -O “sample-metadata.tsv” \
         “https://data.qiime2.org/2019.1/tutorials/moving-pictures/sample_metadata.tsv”
   3. Obtain and import the files
      1. mkdir emp-single-end-sequences
      2. 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”
      3. 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”
      4. qiime tools import \
         –type EMPSingleEndSequences \
         –input-path emp-single-end-sequences \
         –output-path emp-single-end-sequences.qza
   4. Do Demulitplexing Sequences
      1. 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
      2. qiime demux summarize \
         –i-data demux.qza \
         –o-visualization demux.qzv
      3. qiime tools view demux.qzv ( Use this file to view under Qiime 2 View)
   5. Sequence quality control and Feature table construction (DADA 2)
      1. 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
      2. qiime metadata tabulate \
         –m-input-file stats-dada2.qza \
         –o-visualization stats-dada2.qzv
      3. mv rep-seqs-dada2.qza rep-seqs.qza
         mv table-dada2.qza table.qza
   6. Create a Feature table and Feature Data summaries
      1. 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
   7. Taxonomic Analysis
      1. 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”
      2. qiime feature-classifier classify-sklearn \
         –i-classifier gg-13-8-99-515-806-nb-classifier.qza \
         –i-reads rep-seqs.qza \
         –o-classification taxonomy.qzaqiime metadata tabulate \
         –m-input-file taxonomy.qza \
         –o-visualization taxonomy.qzv
      3. qiime taxa barplot \
         –i-table table.qza \
         –i-taxonomy taxonomy.qza \
         –m-metadata-file sample-metadata.tsv \
         –o-visualization taxa-bar-plots.qzv

Conclusion/Future Steps: This lab helped us practice using the moving picture tutorial. It allowed us to see images of graphs and box and whisker plots. The commands from Qiime allowed us to separate the sequences into specific species on the bar graph. It helped us create files so we can view them under Qiime 2 view and see how these files can be manipulated to make the results better.  They can be manipulated by cutting parts of the graphs off if those parts results aren’t good for use. For future steps, when we sequence our ciliate DNA, we will be using Atmosphere and Qiime to separate the sequences to specific groups of ciliates. Using Qiime uses small amounts of data while Atmosphere will hold all the data we have for sequencing and separating our data.

Purpose: The purpose of this lab to be able to learn how to use certain commands from the QiimeWiki and apply them on our apple computer terminal (MacOS). We did this so we could run Qiime once we were able to access atmosphere of Cyverse.

Procedure:

1. Open the terminal of the Apple Computer
2. Go on google and download the “64 bit miniconda .pkg” from https://docs.conda.io/en/latest/miniconda.html
3. Once its down downloading, close the terminal and reopen
4. Type “conda update conda” on the terminal and wait a couple of seconds for it to be running
5. Type “conda install wget” and wait a couple of seconds for it to be installed
6. Type “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”. This should take a couple of minutes to run
7. Type “source activate qiime2-2019.1” to activate the qiime program
8. If no error has occurred in downloading this program, then the program has been successfully installed into the terminal
9. Practice using other commands available for the program and terminal.

Vocabulary Learned:

1.      Qiime: A powerful, extensible, and decentralized microbiome analysis package with a focus on data and analysis transparency.

2.      Demultiplex: performs quality control of sequences through QIIME

3.      Denoise: removes unwanted sequences that don’t match your sequences

4.      Feature Table: defines the logical tree structure of features and contains the columns shown in the table

5.      Alignment: removes certain noise that can affect the sequences. Makes the sequences match.

6.      Phylogeny: the study of evolution, diversity, and the way different organisms and species are related to each other.

7.      Diversity Analysis: Alpha and beta diversity analysis. Applies related statistical test, and generates interactive visualizations.

Commands Learned:

1. used the Say [command] (computer read out what we typed)
2. mkdir letsgetcilli (creates the fil “Letsgetcilli”)
3. ls (allows us to see the list of files in our computer)
4. used the “conda update conda” command (Update the miniconda application)

Conclusion/Future Steps: After completing this lab, we were successfully able to download the Qiime program into our terminal for later use once everybody has access to Cyverse. Running the Qiime program allowed us to access new commands and practiced a couple of those commands and a couple of others that the terminal already knew. We still need to continue to learn how to use Qiime and its commands properly so once we start doing cloud computing , we will know what we are doing when running data. When everyone has access to Cyverse, we will able to start using atmosphere to start cloud computing using the samples from the Chelex and eDNA extraction method.

Objective: The objective of this lab was to be able to understand and learn more about the process of next generation sequencing and Metabarcoding. Another objective we had was to create and account on CyVerse and understand its purpose and what kind of functions this data program has to offer.

Purpose: The purpose of this lab was to be to understand the use of Next Generation Sequencing by reading articles on. The knowledge we gain was used to do a challenge to order the steps of illumina dye sequencing. The purpose of CyVerse is to be able to use this program to compile large amounts of data, in this case we will be collecting large genomic data sets from our Chelex and eDNA extraction procedures. Through this we will be able to determine what kind of ciliates were in our sample by analyzing the sequence DNA in the CyVerse program.

Procedure:

CyVerse: 

1. Make an account
2. write username on spreadsheet

Illumina dye Sequencing Challenge:

1. Arrange the order of the steps in this sequencing

Q/A:

• Tagmentation
  • First step after DNA purification. Enzymes (Transposases) randomly cut DNA into short segments called “Tags”
• Reduced Cycle Amplification
  • Sequences for primer binding, indices and terminal sequences are added. Attached to flow cell
• Bridge Amplification
  • Cluster generation can begin. One reverse and one forward strand is used.
  • The DNA strand bends and attaches to the oligo that is complementary to the top adapter sequence.
  • Happens for thousands of clusters all over the flow cell at once
• Clonal Amplification
  • DNA strands will bend and attach to oligos.
  • This step important for quality control
  • Polymerases will synthesize a new strand to create a double stranded segment. This will then be denatured so DNA from that area come from one source
• Sequence by synthesis
  • Primers attach to the forward strands and a polymerase adds fluorescently tagged nucleotides to the DNA strand
  • One base added per round
  • Machine records what base was added
• NGS Analyzed
  • Sequence occurs for millions of clusters
  • Allows scientists to see the complete sequence even though an unfragmented sequence was never run

Final Poster:

Conclusion/Future Steps:

After this lab, we learned what were the correct steps involving Next Generation Sequencing through illumine dye sequencing. We also made a CyVerse account and learned the functions that we could use for this project. Future steps that we need to take is taking this information that we learned and applying it to our DNA samples that we extracted from Chelex or eDNA. Using this method, we can sequence the DNA we have and compile all the genomic information we have into CyVerse so we can see if were able to sequence any ciliates in our samples. Using CyVerse, we can analyze the data sets that we get when we sequence our sample in future labs.

Objective: Present rough draft of ciliate presentation poster to class

Purpose: The purpose of this is to see what we can improve/change on the poster to get it ready for the final draft and present it at the research seminar at the end of the year.

Procedure:

1. Prepare to present rough draft of poster to class.
2. Meet at computer lab.
3. After presentation, use tips/constructive criticism to revise presentation poster.

Changed:

1. Methods/Pictures
2. Title
3. Introduction
4. Conclusion

Data:

Conclusion/Future Steps: We will improving on the poster the things that need to be changed. The constructive criticism will help us improve on sections that need to be changed. As a group, we will collaborate to things we want to add and change using the suggestions provided from the rough draft presentation and our thoughts. Later the final draft, will be presented at the freshman symposium.

Objective: The objective of this lab was to set up a Gel Electrophoresis procedure so we could run our PCR DNA samples through it.

Purpose: The purpose of this lab was to see if our samples were successfully amplified in PCR by running our DNA samples in our Gel Electrophoresis procedure. We wanted to see if any DNA was present from Chelex or eDNA from soil.

Materials:

• Micropipettes
• Gel Block
• Electrophoresis box
• Power source
• Loading Buffer
• DNA Sample (PCR Sample)
• Kb Ladder

Procedure:

1. Take out gel and grab PCR sample
2. Label the wells for control and sample of DNA
3. Place Gel into Gel Electrophoresis container
4. Fill the container with buffer solution
5. Fill the first well with 5 microliters of Kb Ladder
6. Fill each well with control or sample. Each 10 microliters each
7. Connect Gel Electrophoresis into a power source and let it run for 30 minutes at a 100 volts.

Results:

PCR Gel Electrophoresis

Conclusion:

After seeing the Gel Electrophoresis under the U.V light, the best sample that was clearly detectable was the first sample in well 1. Well 1 was extracted DNA using the Chelex method. The rest of the wells shows very little concentrated DNA or none at all.

Future Steps:

After this lab, we will be using the best DNA samples that showed very concentrated amount of DNA in the Gel Electrophoresis run. One of my partners in the group was able to have a successful amplification of his DNA using extracted DNA from the soil so we will be using his DNA for future metabarcoding procedures. Our samples were not successful besides his.

Objective:

The Objective of this lab was to be able to set up our DNA into a PCR reaction solution using the other components in PCR.

Purpose:

The purpose of this lab was to see up the DNA sample for PCR so it can be run through a thermal cycler to make more copies of our DNA. The thermal cycler will be used to make more DNA in a three step process which is Denature, Annealing and Elongation which is repeated in cycles.

Materials:

DNA Sample:

• 2x taq: 12.5 ul
• DNA: 3.73 ul
• Primer (10 um): 1 ul
• water: 4 ul

Control Sample:

• 2x taq: 12.5 ul
• DNA: 0 ul
• Primer: 1 ul
• water: 11.5 ul

Note: 2x taq- contains the nucleotides, buffer and polymerase.

More Material:

• Pipettes (plus tips)
• Centrifuge
• H20
• DNA sample
• Primer
• 2x taq in tube
• container of ice

Procedure:

1. Before starting the lab, clean the table with 10% bleach solution to prevent samples from being skewed and contaminated for PCR. Clean pipettes also and wear gloves to prevent any DNA containments from getting into sample.
2. Gather material: H20, Primer, DNA sample, 2x taq solution, and microtube. Place them into a small ice container
3. Transfer 12.5 ul of water to a clean microtube using the micropipette
4. Transfer 3.73 ul of DNA sample to the microtube. Centrifuge the sample to mix it.
5. Transfer the DNA/water solution to the 2x taq microtube.
6. Centrifuge sample again
7. Take the sample to ice box and label which box you are.
8. Repeat these steps again for the control sample using the measurements from the control instructions.

Conclusion:

After making the DNA solution for PCR, we put our samples into a ice box so they could be placed into the thermal cycle later. The DNA will be run through the thermal cycle so they could be denature, annealed, and elongated to make more copies of DNA. The prime will be based on the ribosomal 18s RNA for this PCR procedure (V4 primer).

Future Steps:

For next weeks lab, we will be using the DNA that went through PCR and run the DNA through another Gel Electrophoresis experiment to see if PCR worked and if any of our ciliate DNA can be further used for future experiments. All of our samples will be tested again in Gel Electrophoresis including the control solution we made.

Objective:

The objective of today’s lab is to determine if any DNA is in our samples by performing Gel Electrophoresis. After Gel Electrophoresis has been performed, images will be taken of the Gel to detect any DNA and to test the purity of the DNA through the spectrophotometer.

Purpose:

The purpose of this lab is to be able to see if any ciliate DNA had been extracted from our culture using the Chelex procedure. Using the spectrophotometer, we tested how pure the DNA is and take images of the gel to see if any DNA had been detected and if so, how concentrated it is.

Materials:

• Gel with wells
• Electrophoresis box
• Power supply box
• Loading Buffer
• DNA sample
• Spectrophotometer
• U.V light imager
• Micropipettes
• Gloves

Procedure:

Running Gel Electrophoresis:

1. Take DNA out of fridge and thaw out
2. In a microcentrifuge tube, add 9 microliters of DNA and 1 microliter of loading buffer to make up a volume of 10 microliters.
3. Vortex the sample
4. Label the wells for which sample is going in
5. Slowly insert the DNA using a micropipette into well
6. Connect the positive and negative cords to the power source
7. Let it run for 20 minutes at 100 volts
8. Watch the sample to make sure it moves

Using Spectrophotometer:

1. Turn on spectrophotometer
2. When its fully running, add a blank (water) to calibrate the machine
3. After calibration, clean the arm and holder with a wipe
4. Add a small drop of your DNA sample to the machine and run it
5. Record the data seen on screen

Imaging DNA:

1. Place Gel Electrophoresis block onto a slide and into the imagine machine.
2. Take a image under the U.V light and save it on the computer
3. Optional: Use the U.V light. Use glasses when viewing it under the light.
4. Record what you see

Results:

Spectrophotometer Results:

• A260/280
  • 1st run: 126.5 ng/ul
  • 2nd run: 134.1 ng/ul
• Purity: Ideal=1.8
  • 1st run: 1.65
  • 2nd run: 1.68

Gel Electrophoresis under U.V light

Gel Electrophoresis running

Image using scanner

Conclusion:

After doing this procedure, we were able to find DNA in our Gel Electrophoresis procedure when put under U.V light. The amount of DNA in each well was very small for the Chelex samples but it was more concentrated from the eDNA of the soil. The spectrophotometer showed the sample misses the A260/A280 mark and was below the 1.8 mark.

Future Steps:

For the next part of our research, since we found DNA in our sample, we will be performing PCR to amplify the DNA sample. After performing PCR, we will possibly be testing the Gel Electrophoresis again to see what our samples look like again with more concentrated DNA.

Objective: The objective of this week is to practice DNA puritifcation on our soil samples, set up Gel Electrophoresis gel and write down the methods to our research in scientific format.

Purpose: The purpose of this lab is to be able extract and use purified DNA from our culture or soil samples and run them through the Gel Electrophoresis.

Materials:

• Chelex sample
• Microwave
• Flask
• D.I water
• Vortex
• Centrifuge
• TAE buffer
• Agarose
• Ethidium Bromide
• Electrophoresis Box

Procedure:

Gel Electrophoresis: 

1. Make 1 Liter of 1xTAE using 4mL of TAE solution with 36 mL of D.I water and add 0.4 grams of agarose. Swirl gently in a flask
2. Heat up the solution in the microwave for 2 minutes. When its finished gently swirl it again.
3. Let it cool for a couple of minutes before proceeding to the next step.
4. Add 0.002 microliters of Ethidium Bromide to the solution to make it a 0.5 ug/mL concentration in the solution. Swirl gently
5. Set up the gel electrophoresis box with rubber bumpers and then add comb to make the wedges in the gel.
6. Add the solution to the box
7. Wait half an hour so the solution can solidify
8. After the solution solidifies, add a small amount of 1xTAE buffer over the Gel.
9. Place the gel electrophoresis box in a plastic bag and leave it in the fridge.

Continue Chelex Extraction:

1. Boil the sample in a water bath for 8 minutes in 100 degrees Celsius.
2. Vortex the sample for 1 minute
3. Centrifuge the sample for 3 minutes at 16000 g
4. Transfer the supernatant in microcentrifuge tube. Throw away pellet
5. Properly label your tube with soil identifier.

Conclusion: After this lab, we were able to make a gel mold for our Gel Electrophoresis and to finish extracting DNA using the Chelex protocol. Out of the group, two of us did the Chelex protocol and the other did the DNA extraction from soil using the Purification protocol.

Future Steps: In our next lab, we will be using our extracted and purified DNA sample on our Gel Electrophoresis lab. We will run the DNA in the gel and see what our DNA length looks like after we stain the DNA with dye and observe it using U.V light.