Lab 14: Poster Presentation and Abstract Submission

The purpose and objective:

The objective of this lab is to complete the metadata form, final draft abstract and scientific poster.

Date: 04/26/2019

Procedures:

1. Fill in the form of soil metadata through Excel;
2. Discuss and write the abstract, and edit the scientific poster;
3. Upload file on Canvas and box.

Data:

• Metadata

• Abstract

Isolation and Purification of Ciliate DNA from Ilex Vomitoria

Nearly 85% of ciliate diversity has not been accounted for, yet ciliates play a crucial role in soil ecosystems, ensuring the cycling of materials and energy flow of other microorganisms and serving as predators regulating the environment. This study was conducted to develop a well-defined methodology that could be used to explore soil ciliates on a molecular level via sequencing. Soil samples were taken from an Ilex vomitoria and DNA was extracted using the silica bead extraction method. Metadata analysis of the soil showed that the soil type was sandy clay loam and had a pH level of 6.5. Gel electrophoresis showed that crude DNA was extracted from the soil samples and PCR was able to amplify an 18S V4 region of the DNA. From the Nanodrop analysis, performed after the extraction of a crude DNA sample and DNA purification, the extracted DNA had a purity of 1.46 (A260/A280), which is the expected score of a pure sample. The utilization of this less costly and more efficient protocol can yield DNA samples of a good purity that can be used for future sequencing and taxonomic identification of organisms within a collected sample.

• Poster

Conclusion and The future goal:

In this lab, based on discussion, suggestions and comments, we edited and finished our final abstract and poster successful. In the future, we will do a presentation of our scientific poster next week and I am looking forward it!

Lab 13: Soil eDNA Metabarcoding Analysis: Qiime2

Purpose and the objective:

The purpose of this lab is use QIIME2 to analysis soil eDNA from eDNA sample extracted from out soil sample and the rhizospehere of Bermuda grass (from the Blackland Prairie Soil).

Date: 04/18/2019

Procedures:

1. Download the folder
2. Reinstall the terminal
3. Turn on the terminal and change directory to the downloaded folder
4. Activate QIIME2 in the terminal
5. Following the steps on the doc with QIIME2
6. Record the data and analysis and complete QTM

Data:

Conclusion and the future goal:

In this goal, we complete the procedures and analysis successfully. From the level 2 of taxa bar plot, we could get that there are 24.864% matching to SAR, 35.451% matching to Opisthokonta, 23.805% matching to Archaeplastida for soil. 1PS. eDNA. And there are 54.084% matching to SAR, 26.358% matching to Opisthokonta, 0.706% matching to Archaeplastida for soil. 1C. eDNA. Also, from performing the BLAST, the sequence analysis of ciliate is close to Paramecium caudatum. From the QIIME2, we can clearly know the relative abundance of species in a short time. In the future, I hope to build a more complete analysis system to analyze our soil diversity and finish our presentation successfully.

Week 12: UNIX and Setting up the System

The purpose and objective:

The purpose of this lab is to learn the different illumine file types and complete the Moving Pictures Tutorial, and learn different graph including quality plots graph, metadata graph, DADA2 graph, Taxa Plot.

Date: 02/11/2019

Procedures:

1. Reinstall the Qiime2 and test it with command “source activate qiime2-2019.1”;
2. Type pwd to show the user name;
3. Type this before beginning the tutorial
4. Sample metadata with type this wget 
5. Obtaining and importing data via type this
6. Select these download option
7. Type this to get emp-single-end-sequences.qza
8. Demultiplexing sequences
9. Type this to get demux.qzx
10. Choose option 1: DADA2 and type command to get the stats-dada2.qza and table-dada2.qza and rep-seqs-dada2.qza
11. Type this to get stats-dada2.qzv
12. Type this to get rep-seqs.qza ad table.qza
13. FearureTable and FeatureData summaries, type this to get table.qzv and rep-seqs.qzv
14. About Taxonomic Analysis, type these
15. Drag all qzv. Document to qiime 2 view and got the graphs.

Data:

• Demultiplexed sequence counts summary
• Interactive Quality Plot
• Sequencing Table
• DADA2 Table
• Taxa Bar Plot
• Taxonomy Table
• Moving Picture Sample Metadata

Conclusion and the future goal:

In this week, we reviewed our research goal, illumine sequencing and reinstalled the QIIME2. Also, we learned FASTA and FASTQ, and finished the procedures of moving pictures tutorial successfully. Moreover, we got some -.qzv documents through the tutorial procedures, and dragged them to the Qiime 2 View that occur including quality plots graph, metadata graph, DADA2 graph,Taxa Plot, and then we analyzed them. In the future, I hope to learn more knowledge about Qiime2 to analysis our sample.

 Week 11: Poster Presentations (SW) Cloud Computing and Jupyter Notebooks

Lab10: Next Generation Sequencing and Metabarcoding

The purpose and objectives:

The objective of this lab is learning the procedures of Illumina sequencing and set up the CyVerse account.

Date: 03/28/19

Procedures:

1. Learn the Illumina procedures from PowerPoint;
2. Rank the work cards with group member;
3. Complete QTM (including draw the picture of Illumina sequencing);
4. Add Username of CyVerse on the Excel;

Data:

Conclusion and The Future Goal:

In today’s lab, we rank the procedures and learn about the next generation sequencing. Also, we learn the function of CyVerse and how it helped our experiment. In the future, I hope to learn more about our sample and soil ciliates diversity by learning different analysis methods.

Lab 9: Poster Presentation

The purpose and objective:

The objective of this week lab is to present the rough draft of the science poster on the computer lab and receive the feedback with the goal of improving our ability to make a poster.

Date: 03/21/2019

Procedure:

1. Prepare the rough draft;
2. Do a presentation on the computer lab;
3. Receive feedback;
4. Discuss and improve it.

Data:

Conclusion and the future goal:

In this lab, we do a presentation about our rough draft and get constructive criticisms of our post. About the criticisms, we need to add the graphs with data and label it, and combine the materials and methods instead of separating them. Also, we need to add information on conclusion, results and references, and change the organization of the poster. After this lab, we will add information on the poster. For the future goal, I hope that we could improve our Discussion section on the poster with references or other studies.

Lab 8: PCR results; Scientific Poster Design

The purpose and objective:

The purpose of this lab is to run the DNA sample through gel electrophoresis and analyze the image. Also, we take the midterm exam and write the draft of poster.

Materials: kb ladder, DNA sample, agarose gel. Gel electrophoresis, voltage machine, micropippetor.

Procedures:

1. Add 5μl of 1kb ladder in well #1;
2. Add 10μl of control prepared sample in well #7;
3. Add 10μl of prepared DNA sample in well #8;
4. Run the gel at 100V for 30 minutes;
5. Get the image of gels.

Data:

Conclusion and the future goal:

In today’s lab, we analysis our DNA sample to see if enable to perform PCR. From the image, we could see the clear V4 band that indicates we have a high-quality DNA sample. Also, we could learn that the amplified DNA showed approximately 400-500bp. Moreover, we wrote the poster draft. In the future, I hope that we could finish our poster successfully and learn more about the diversity of ciliates in the soil ecosystem.

Lab 7: PCR amplification of DNA

The purpose and objective:

The objective of lab is setting up the Polymerase Chain Reaction (PCR) by using our DNA sample to amplify a piece of DNA sample and discussing the scientific poster design.

Date: 02/28/19

Materials: DNA sample, 10% bleach, ice box, D.I. water, tube, primer, 2X Taq Mix, micropipettor, pipette tip

Procedures:

1. Wear gloves and clean the desk with 10% bleach;
2. Place an empty tube, water, DNA sample, and V4 Primer in a box with ice;
3. Add 9µl water first and add 9µl DNA sample to create the 1:10 dilution and mix the solution with micropipettor;
4. And then, take two tubes which have already containing 12.5µl 2X Taq Mix;
5. Label the control and treatment;
6. For the control tube, add 1µl primers (10µM) and 11.5µl water and mix them;
7. For the treatment tube, add 1.7µl DNA sample, 1µl primers (10µM), 9.8µl water and mix them;
8. Make sure close all tubes and pipette tip boxes when not in use.

Data:

Storage:

We put the DNA sample, water, primers and tubes back to the box with ice.

Conclusion and the future goal:

In the lab, we learned lots of information about PCR and set up PCR by using our DNA sample successfully. Also, discussed about the outline of Poster. Moreover, we will run Agarose Gel and PCR one more time in the future. And I hope that we could analyze our DNA sample through PCR and learn more about the diversity of ciliates.

Lab 6: Gel electrophoresis and DNA Analysis

The purpose and objective:

The objective of lab is determining if there is any DNA in our sample through running the gel electrophoresis and using the Nanodrop to measure the level of absorbance. Also, we use the CCD system to capture the DNA image.

Date: 02/21/19

Procedures:

• Running the Agarose Gel:

1. Placed the gel in the gel box and turned the gel to the position that furthest away from positive electrode;
2. Added 1X TAE buffer to cover the gel;
3. Removed the border of gel;
4. Mixed 9µl of sample and 1µl 10X loading buffer into a microfuge tube, and then spin the tube;
5. Load the 10µl of one sample in well 1 and load another sample in well 7;
6. Load the 5µl of DNA Mass STD #1 (500ng) in well 3 and load 5µl of DNA Mass STD #2 (15ng) in well 5;
7. Run the gel at 100 voltage for 20 minutes.

• DNA Analysis – Nanodrop/Imaging

1. Took sample to the imaging lab;
2. Added 1µl water to the Nanodrop and clean it
3. Added 1µl sample to the Nanodrop and waited the results showing on the screen;
4. Put the gel on the MBC C305 plate to capture the image and recorded it.

Data:

• Nanodrop

• Imaging

Conclusion and the future goal:

In today’s lab, we put 50µl DNA Mass STD #1 (500ng) into the well 3, which is darker in the image; however, we could analysis the DNA sample mass between 15-500 ng. In the future, I hope that I could run PCR methods successfully and study the diversity deeply through our sample.

Lab 5: DNA Extraction Part 2

The purpose and objective:

The objective of this lab is purifying our DNA sample that extracted on the last week and creating Agarose Gel Eletrophoresis.

Date: 02/14/29

Materials: sterile water, tube, warm DNA resin, syringe barrel, vacuum filtration manifold, micropipettor, isopropanol, agarose powder, Erlenmeyer flask, microwave, Ethidium bromide, gel electrophoresis box, plastic bag, freezer, gloves, stock solution.

Procedures:

• DNA Purification:

1. Take the “crude soil DNA” extraction and add sterile water to 1ml line.
2. Transfer this 1ml sample to a 15ml tube. Add 2ml warm DNA resin and mix by inversion several times.
3. Assemble a column bottom on syringe barrel and place it on the vacuum filtration manifold.
4. Add half of DNA liquid to the column and add remainder of sample once half of liquid has been pulled through.
5. Wash the column by adding 2ml 80% isopropanol and using the vacuum to pull it through.
6. Add a few isopropanol to allow it flowing and repeat three times.
7. Transfer the column from the barrel and place it in a 1.5ml tube.
8. Spin at 8000xg for 5 minutes and put it in 80°C+ heat block for 30s-1min.
9. Place column in new tube and add 50µl sterile diH2O heated to 80°C directly.
10. Incubate 1 min and spin at 8000xg for 1 min as well.
11. Label the tube correctly and place in freezer.

• Agarose Gel Elctrophoresis:

1. Add 4ml of 10x TAE stock solution, 36ml D.I. water and 0.4g agarose powder to Erlenmeyer flask and swirl it until mixed.
2. Microwave the solution for 1 min and swirl it for 1 min as well.
3. Add 2µl of Ethidium bromide to solution and mix the solution.
4. After cooling, set up a gel electrophoresis box and transfer the solution into the mold until reaching the top of mold.
5. Put the mold into a plastic bag and place it in the refrigerator.

Conclusion and the future goal:

In this lab, we purified the DNA sample that we did on the last lab and create Agarose Gel Eletrophoresis successfully. And I hope that we will test out DNA sample on next lab successfully as well. Also, in the future, I hope that I can learn more skills on DNA extraction and learn more knowledge about ciliates.