Abel Thomas

4/26/19

Purpose/Objective:

The purpose of this lab was to label and understand our metadata along with correcting our poster and our perfecting the final abstract for the poster.

Procedure:

1. Vote on a logo
2. Finish total metadata for the class
3. Revise poster and the abstract

Metadata:

Title:

The Observation of Biodiversity on the Baylor Campus through Metabarcoding

Abstract:

Soil biodiversity plays an integral role within the soil microenvironment as well as the sustainability of the macroenvironment. As a field experiment, soil was collected and analyzed from the rhizosphere of Quercus macrocarpa for metabarcoding using the silica bead DNA extraction method. Using this protocol for DNA extraction, a positive PCR product with relatively pure eDNA confirms the presence of soil microorganisms, possibly ciliates. After collection, extraction, purification, gel electrophoresis, amplification of DNA using PCR, and gel electrophoresis of the amplified DNA, next generation sequencing and analysis are ready to be conducted by quantifying the diversity present through the QIIME2 application and basic bioinformatics tools. By quantifying the microorganisms present, we can better learn and understand the optimum environments for ciliates and other microorganisms for future application. Further testing is necessary to better understand the extent of biodiversity present in the soil of the rhizosphere on Baylor campus. Bioinformatics analysis will be further conducted in regard to quantifying and characterizing qualitatively the importance of biodiversity in the environment.

Poster:

Storage: After inputting the metadata, we went up to the computer lab and fixed up our poster and our abstract to be submitted into Box.

Conclusion/Future Goals: In lab we were able to complete our metadata for our entire class. And then using the computer lab we were able to complete and fix our errors in our abstract and our posters. With this information we would be able to send our information to a lab and eventually use QIIME to analyze it.

Abel Thomas

Section 21

4/18/19

Purpose: The purpose of this lab was to continue with the qiime2 procedure and help us see the difference in the soil and the DNA found from both.  We were able to use more coding to help us see the exact difference in the barcodes of both soil types.

Purpose:

1. First we opened up terminal and made sure we still had qiime, if we did not then we redownloaded it
   1. Use “source activate qiime2-2019.1” to start qiime
2. Download the CILICURE_2018 folder from the Box which is linked in the powerpoint in the module section
3. Copy the folder from your Finder by double clicking
4. Change directory by using
   1. “cd /Users/abelthomas/Downloads/CILICURE_2018”
5. Add folders and sequences using the code
   1. qiime tools import \–type EMPPairedEndSequences \–input-path emp-paired-end-sequences \

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

6. Then we do demultiplexing which separates each barcode into its own
   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

7. Then we denoised to 220 so we could make sure the DNA was small but still significant to see that there is a difference between the 2. Denoising cuts the sequence to the amount you said it to.
   1. 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

      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

8. Then we created a phylogenetic tree to help show the relation between the organisms.
   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

9. After that step, we specified the phylogenetic tree by making files to organize the tree into the taxa and help show the concentrations of each organism in the soil.
   1. 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/Future Goals: After all these steps were completed then we were able to drag all the qzv files over onto Qiime2 View and see what the files showed us, the biggest one was the taxanomic bar plot which showed us the concentrations of ciliates found in each soil type. Then we were able to view all the sequences use the rep seqs qzv where we picked our sequence and ran it through BLAST and then found a PubMeds article on it. For the future we could use this for our later work with our own soil samples and find what we see from that.

Abel Thomas

4/4/2019

Purpose/Objective:

The purpose of this lab was to learn commands for terminal and helped download Qiime. Qiime will be used to analyze DNA sequences.

Procedure:

1. Open terminal on the Mac computer. Dell computers do not work
2. Download the files needed for Qiime using the intial code from the Qiime website
3. Download miniconda 64 bit (pkg) folder from the proper website
4. To let conda update you must type “ conda update conda” into the terminal, this will get you the right version
5. Type “conda install wget” into terminal to allow it to properly install on the mac
6. The next command we used in terminal is “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 helps download the qiime properly
7. Allow for the downloads to take place, wait for the program to download
8. Use the program to view images for the lab.

Commands:

Learned how to have the computer say something back to you “say + words”

how to look through my directory “cd desktop” will show everything that is on your desktop

how to grab something use wget “wget Applications” will bring you all the files in your Applications file

“conda update conda” will update conda so it fits in your terminal

“Source activate qiime2-2019.1” makes your qiime work properly

Conclusion:

We were able to download qiime onto the Mac as well learning to understand more about the Terminal and how to use commands to browse through our computers. We were able to start up qiime and start running it so we will be able to use it for the upcoming weeks. The terminal ran through the commands that we copied and put into it so qiime will be operational.

Abel Thomas

3/28/19

Objective: During this lab, students became more hands on with the aspect of next generation sequencing.

Procedure:

1. Get with a partner and make sure the cards that were given to us were put in order. Taylor will come and check to see if it is correct. If not, then students will fix until it is right.
2. Understand the process of next generation sequencing by filling out the QTM with the partner
3. Listen as Dr. Adair explains and shows the benefits of Atmosphere and how we can use it as we understand perfection.

Conclusion/Future Goals:

As today wrapped up we were able to have a better understanding of the new form of sequencing and how it works to its core. It was interesting to see how much detail and how microscopic the changes are and how much of an affect that can have. The future goal is to hopefully let the advisors see which data and DNA to choose and see a company use this method and do our own form of sequencing and analyzing the information that we get.

Storage: We made sure we logged off our computers and handed back the colored pencils as well as the baggie with the cards.

Abel Thomas

3/21/19

Objective:

The objective of this lab was to help the students have a better understanding of presentation skills as well as poster making.

Purpose:

We observed other students present and saw what we needed to fix within our own poster. Our poster will have more information and data but as of now it was a very basic and rough draft. We will include consistent fonts and pictures that better show our information and make sure our poster looks more presentable rather than just being so factual. We will also make sure that we know our topics and if we do not know something we will say that we do not know it.

Conclusion/Future Goals:

We will meet up later this week to perfect our poster and our presentation. We have a lot of things to fix but it will be easy to fix since we got a lot of constructional criticism. Our poster will include more information and have references as well.

Biology Poster (1)-vlxqb6

Abel Thomas

3/8/19

purpose: The purpose of today’s lab work was to examine the work of the PCR that was added last week. We did this through gel electrophoresis.

Materials:

Agarose Gel

Control sample

Treatment sample

Micropippeter

Electrophoresis container

Procedure:

1. Take samples, both control and treatment from the front of the room
2. Bring back to desk and get micropippeter set for 10 uL
3. Make sure agarose gel is in the electrophoresis container
4. Micropippete the samples into the wells, making sure the wells are on the negative side
5. Set the machine to 100V for 30 minutes and wait
6. Observe the results

Conclusion/Future Goals: Our DNA was shown to have a lot of sample in it thanks to the PCR. That means we had a very concentrated amount of DNA in that specific region.This will help us when we send off our DNA for sequencing to see what type of organisms were in there. After that we will be able to fully complete our posters.

Storage: We made sure to grab all of our belongings and also made sure that the machine was running properly when we left.

Abel Thomas

3/1/19

Purpose:

The purpose of this lab is to set up the PCR reaction using our DNA and combining it with the Supermix along with other substances. We also worked on our poster design.

Materials:

gloves

eDNA

primers

Supermix

water that was autoclaved

PCR tube

Falcon Tube

Bleach

Micropippete

Procedure:

1. Put on gloves
2. bleach the table to ensure clean working environment
3. calculate the amount of DNA and water needed to make sure that both the control and treatment solutions end up being 25 uL
4. obtain an ice bath along with an empty tube, the tube containing DNA, and the V4 primer tube
5. obtain 2 tubes that contain the supermix, label which one will be the control and the treatment
6. add 1 uL of primer to both tubes
7. Add 11.5 uL of water to control and 10.12 to the treatment
8. Add 1.38 uL of DNA to treatment tube
9. make sure that both tubes are mixed well

Storage:

We made sure that we cleaned our work area and put our tubes in the ice container to preserve them. We also properly removed the pippetes.

Conclusion/Future Goals:

We were able to set up our DNA for PCR amplification. Next week we can hopefully observe the PCR amplification and then the week after we can send our information to get sequenced and see the DNA that we took from the soil.

Abel Thomas

2/21/19

Purpose/Objective: The purpose and objective of this lab is to be able to see what type and how much DNA we have in our samples.

Materials:

Nanodrop

1X TAE buffer

31ng DNA mass

125ng DNA mass

250ng DNA mass

500ng DNA mass

10X leading buffer

Agarose Gel

Electrophoresis Gel

Nanodrop

DNA specimen

Procedure:

Gel Electrophoresis:

1. Remove the gel from the mold and transfer it onto the transfer dock and cover the gel and the rest of the docker with 1X TAE buffer
2. Select the walls in which the DNA will be put into
3. Extract 9 uL of DNA and transfer it into eppendorf tube
4. Add 1 uL of 10X loading buffer
5. mix solution
6. Add 10 uL of DNA from your DNA sample into the second well
7. Add 5 uL of DNA 31 ng mass to 5th well
8. Add 5 uL of DNA 125ng mass to the first well
9. Add 5 uL of DNA 500 ng mass to the third well
10. Run the agarose gel at 100 V for 20 minutes
11. Take the gel to the UV illuminator and observe the image to see how much DNA was found in your sample

Nanodrop:

1. Clean the machine and ensure the machine is clean and dry
2. Place 1 uL of DNA sample on the top of the holder
3. Close the machine and wait for results
4. Analyze the results

Results:

Our DNA ended up being 722.3 ng/uL with 1.56/1.8 for the A260/A280 ratio and 1.14/2 for A260/A230 ratio. Our DNA peaked at 260 but with these numbers we can say we still had some proteins and carbohydrates in our sample which showed that our data was not pure.

Storage: We made sure to throw the gel away in the biohazard bag and made sure to rinse and dry everything carefully to ensure there was no contamination. After we did that, we were able to wipe down our lab area and made sure it was clean and good for the next group.

Conclusion/Future Goals: We were unable to get our sample to be pure DNA but that is okay because we were able to observe the techniques and protocol needed to observe the DNA we retrieved form our soil. Our future goal is to see how our results will end up compared to everyone else’s results. We will use PCR to identify and amplify the DNA we have and then later send it for it to be sequenced.

Abel Thomas

2/14/19

Objective: Today’s lab objective was to assemble all the necessary components we would need to sequence and observe the DNA we collected from our soil.

Materials:

Ethidium bromide

Resin

125ml Erlenmeyer flask

falcon tubes

15 mL test tubes

agarose gel

column

syringe barrel

80% isopropanol

gel mold

comb

Procedure:

DNA Extraction:

1. Make sure the falcon tube contains 1 mL of DNA, if not, add D.I water
2. Add to a 15 mL test tube
3. Add 2 mL of resin into solution and mix by inverting it multiple times
4. Set up a column and syringe barrel on the vacuum filtration manifold
5. Add half  of the solution to the column until it gets sucked through, then put in the other half
6. Wash the column by adding 2 mL of 80% isopropanol and use the vacuum to pull it through
7. Repeat the washing step over again so it rinses clearly
8. Remove the column from the barrel and put it into a clean 1.5 mL tube
9. Spin the column in a centrifuge for 5 minutes to rid of the rest of the isopropanol
10. Put the column into the heat block for 30 seconds to a minute.
11. Put the column in a new 1.5 mL tube and add 50 uL of D.I. water heated to 80 degrees Celsius directly to the column.
12. Incubate for a minute and then spin again in the centrifuge for a minute

Agarose Gel:

1. Measure .4 grams of the agarose powder on a balance
2. Put it into a 50 mL test tube and add D.I. water so it would reach the 40 mL point
3. Shake the tube by inverting it
4. Pour the solution into a 125 mL Erlenmeyer flask
5. Microwave and heat the solution for a minute or until the agarose dissolved
6. Let the solution cool and add 1omg/mL Ethidium Bromide and swirl the final solution together
7. Place the solution into the gel electrophoresis mold
8. Place comb correctly by making sure blue is facing away from the major area of the mold
9. Let the solution cool until it is solidified
10. Remove the comb

Storage:

We made sure we wiped down the table and disposed of all the unnecessary materials. We also made sure to wash and rinse all of the beakers that were used.

Conclusion/Future Goals:

We were able to successfully set up our agarose mold and make sure we isolated our DNA so it would be ready for electrophoresis next week. Our future goal is to use what we used this week and our prior knowledge to use the gel electrophoresis next week to help show which DNA is the best to be sequenced.

Abel Thomas

2/7/19

Purpose: During this lab time, we started using metagenomics to get the DNA from the tree and the soil. We will sequence this DNA and also we will find the ciliates from the soil.

Materials:

Petri Dish

Eppendorf Tubes

Soil Sample

Dissecting Microscope

Compound Microscope

Silica beads

DNA extraction buffer

Pestle and Mortar

Heat Block

Falcon Tube

Ruler

Charcoal

Procedure:

1. We observed the leafs under the dissecting microscope to see if we could find anything that we couldn’t see from our first observation
2. Observe the characteristics of the leaf
3. Get falcon tube from the previous lab
4. Measure the soil, clay, and silt
5. Add 1 gram of the dry soil from the small plate and 1 gram of silica beads and grinded it with mortar and pestle for 5 minutes… Bassam’s soil was chosen because it had the most ciliates that were the best to see
6. Continue to grind with the mortar and pestle and add 10 mg of charcoal, after a minute add 2 mL of the DNA extraction buffer and keep grinding the mortar and pestle
7. Take pippete and put mixture from mortar and pestle into eppendorf tube
8. Put the eppendorf tube into the heat block for 10 minutes at 65 degrees Celsius
9. Spin the eppendorf tube in a vortex for 5 minutes
10. pippete supernatant from eppendorf tube without getting any soil

Data:

There was 1 cm of sand, .8 cm of silt and .3 cm of clay. which means there was 48% sand, 38% silt and 14% clay. this makes our soil to be sandy loam

The leaf was oblonceolate, an obtuse top, a rounded bottom, entire edges, odd pinnately compound

We determined the tree was a Texas Red Oak or Quercus Buckleyi

Storage: We put all our instruments in their appropriate spots and made sure to wash and clean our area and the instruments

Conclusion/Future Goals: We were able to move farther in the metagenomics process. We were able to extract the DNA. Our future goal is to sequence the DNA and help us idetnify the trees and the ciliates around the trees.