# Nanopore for Educators Workshop — Guam

*March 4-5, 2024*
*John F Kennedy High School, Tamuning, Guam*

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Supported by grants from the National Science Foundation (NSF) and National Institutes of Health (NIH):

- NSF Improving Undergraduate STEM Education (#1821657, #2216349)
- NSF Advanced Technological Education (#1901984)
- NSF Arecibo Center for STEM Education and Research (#2321729)
- NIH Science Education Partnership Award (#5R25GM137355)
- NIH Science Education Partnership Award (#5R25GM137355)
- NIH STEP-UP/NIDDK (#5R25DK132965)

Equipment and supplies provided by Oxford Nanopore.

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<img src="https://lh3.googleusercontent.com/d/10x3lQdQo3OWnLLk7RbRMxbM5j4R5TvLU">

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## Instructors

- Jason Williams, Cold Spring Harbor Laboratory, [e-mail]
- Brittany Johnson, Cold Spring Harbor Laboratory, [e-mail]

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## Attendees

Please enter your name, school, (student or teacher), and email address

## Monday March 4th

1. *Hugh Kent*, John F. Kennedy, (Student), [e-mail]
2. Andrew Kang, John F Kennedy, (Student), [e-mail]
3. Bianca Elise Borja, John F. Kennedy, Student, [e-mail]
4. Sidney Savares, John F Kennedy HS, Student, [e-mail]
5. Jenna Sharma, John F. Kennedy High School, Student, [e-mail]
6. Hanna Leona Reyes, John F. Kennedy High School, Student, [e-mail]
7. Nichelle Torcelino, John F. Kennedy High School, Student, [e-mail]
8. Christine McCormic, St. John's, Teacher, [e-mail]
9. Dylan Rasco, John F. Kennedy, Student, [e-mail]
10. Collette P. Trujillo, John F. Kennedy, student, [e-mail]
11. Stephen Medrano, John F. Kennedy, student, [e-mail]
12. Yu-Ying Chen, John F. Kennedy, Student, [e-mail]
13. Mikhayla Marquez, John F. Kennedy, Student, [e-mail]
14. Carolyn Haruo, John F. Kennedy, Teacher, [e-mail]
15. Aidan George (Student), John F. Kennedy, [e-mail]
16. Alan Vitug, John F. Kennedy, student, [e-mail]
17. Anna Rice, John F Kennedy, student, [e-mail]
18. Leonard-John L. Ventura, JFKHS, Teacher, [e-mail]
19. Jeniffer Schumacher, JFKHS, Teacher, [e-mail]
20. Leah M. Medrano, JFKHS (Teacher) [e-mail]
21. Leynebel Senodo, JFKHS [e-mail] (teacher)
22. Crucita Rosero, JFKHS(student), [e-mail]
23. Zena Jun, John F Kennedy HS, student, [e-mail]
24. Elaine Macalinao, THS, Teacher, [e-mail]
25. Crystal Bunoan, Simon Sanchez High School, [e-mail] (teacher)
26. Anita Balmeo, JFK, Teacher, [e-mail]
27. Colette Beausoliel, JFKHS, [e-mail], Teacher
28. Garrett Mangosong, SPCS, Student, [e-mail]
29. Sanjay Sharma, JFKHS, [e-mail], Teacher
30. Gilbert Mangosong, JFKHS, Teacher, [e-mail]
31. Jason WIlliams (CSHL)

## Tuesday March 5th

Please enter your name, school, (student or teacher), and email addres

1. Aidan George, John F. Kennedy, student, [e-mail]
2. Hugh Kent, John F. kennedy, student, [e-mail]
3. Yu-Ying Chen, John F. Kennedy, Student, [e-mail]
4. Crystal Bunoan, Simon Sanchez High school, teacher, [e-mail]
5. Nichelle Torcelino, John F. Kennedy High School, Student, [e-mail]
6. Camia Erika Wy, John F. Kennedy High School, Student, [e-mail]
7. John Paul Lapid, John F. Kennedy HS, Student, [e-mail]
8. Gregoria Rondilla, Okkodo High School, Teacher, [e-mail]
9. Christine McCormic, St John's, teacher, [e-mail]
10. Stephen Medrano, John F. Kennedy, Student, [e-mail]
11. Hanna Leona Reyes, John F. Kennedy High School, Student, [e-mail]
12. Leah Medrano, JFKHS, Teacher, [e-mail]
13. Anna Rice, John F Kennedy HS, student, [e-mail]
14. Bianca Elise Borja, John F. Kennedy, Student, [e-mail]
15. Carolyn Haruo, John F. Kennedy, Teacher, [e-mail]
16. Dylan Rasco, John F. Kennedy, Student, [e-mail]
17. Leynebel Senodo, JFKHS, [e-mail], teacher
18. Elaine Macalinao, THS, Teacher, [e-mail]
19. Andrew Kang, John F Kennedy HS, student, [e-mail]
20. Jenna Sharma, John F Kennedy HS, student, [e-mail]
21. Tricia Togawa, OkHS, Teacher, [e-mail]
22. Colette Beausoliel, JFKHS, teacher, [e-mail]
23. Samantha Torcelino, JFK, student, [e-mail]
24. Anita Balmeo, JFK, Teacher, [e-mail]
25. Sanjay Sharma, JFKHS, Teacher, [e-mail]
26. Garrett Mangosong, SPCS, Student, [e-mail]
27. Leonard-John L. Ventura, JFKHS, Teacher, [e-mail]
28. Dianne Hope T. West, OHS, Teacher, [e-mail]
29. Zena Jun, John F Kennedy HS, student, [e-mail]
30. Franchesca Sarmiento , JFKHS Student, [e-mail]
31. Mikhayla Marquez, JFKHS, Student, [e-mail]
32. Alan Vitug, JFKHS, Student, [e-mail]
33. Crucita Rosero, JFKHS, Student, [e-mail]
34. Jeniffer Schumacher, JFKHS, Teacher, [e-mail]

## Agendas

**Monday**

- Introductions
- Nanopore and 16s background
- Pipette practice
- 16s Shoe-ome experiment
- Swab collection
- Microbe DNA extraction
- PCR

**Tuesday**

- Introductions
- Nanopore and 16s recap
- DNA Barcoding background
- DNA Barcoding - plants
- Chelex DNA extraction
- PCR
- Electrophoresis
- 16s Shoe-ome experiment
- Electrophoresis of 16s samples
- Sequencing prep and loading
- Data analysis
- DNA Learning Center Resources and Curricula

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## Resources

Videos and Animations
- [Intro to microbiome](https://youtu.be/6564K4-_DBI?si=7nU98W79wl-mQVa0)
- [How Bacteria Rule Over Your Body – The Microbiome](https://youtu.be/VzPD009qTN4?si=9Rm3GLLtzHOtYdol)
- [16S rRNA gene sequencing](https://youtu.be/cRLlrzh7lRI?si=4bSxWvBLF7M43aMt)

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## Protocols

### Swab Collection

**Materials**

- Swab collection and DNA preservation tube
- Sterile cotton swab

**Collection protocol**

1. Obtain a shoe you wish to sample. The shoe does not need to be visibly dirty; however, a shoe worn daily is likely to have greater microbial diversity than one that has never been worn.
2. Carefully unscrew the cap of the collection tube without spilling the contents. Next, carefully remove the sterile swab from its package, being cautious not to touch the swab or contaminate it in any way. Dip the swab into the liquid in the collection tube.
3. Use the moistened swab to gently rub the underside and sides of the shoe. A moist swab is more effective at collecting microbes that may be present on the shoe. Ideally, the swab will visibly change in appearance as it picks up dirt and microbes.
4. Return the swab to the collection tube. The swab has a "score," a thinned section of the plastic that allows you to break the swab in half, with the half remaining in the tube fitting just enough to be enclosed.
5. Replace the cap on the collection tube, ensuring that the tube is fully closed and leakproof. The solution in the tube deactivates any microorganisms and preserves DNA. Label the tube with your name and date.

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### Norgen Microbiome

The full formal protocol is available on the Norgen website: [Microbiome DNA Isolation kit](https://norgenbiotek.com/product/microbiome-dna-isolation-kit).

### Materials

- Preserved DNA sample from swab
- Lysis Additive A
- Binding buffer I
- 70% Ethanol
- Binding Buffer B
- Wash Solution A
- Elution Buffer B
- DNA spin columns
- 65˚C water bath
- Ice
- 1.5ml tubes
- Centrifuge

**Protocol C**

#### Part I - Preparing swab sample

1. Add 100 μL of Lysis Additive A to the swab collection tube and vortex briefly.

2. Incubate the swab collection tube at 65˚C for 5 minutes.

3. Carefully remove the swab from the collection tube.

4. Label a microcentrifugue tube and transfer up to 1 mL of the preserved sample to the microcentrifuge tube.

5. Centrifuge the tube for 2 minutes at 20,000 × g (~14,000 RPM). A
thin white layer will form on the top of the supernatant.

6. Label a new microcentrifuge tube and carefully transfer 700 μL of
supernatant, without the white layer debris, to the microcentrifuge tube.

7. Add 100 μL of Binding Buffer I, mix by inverting the tube a few
times, and incubate for 10 minutes on ice.

8. Spin the lysate for 2 minutes at 20,000 x g (~14,000 RPM) to
pellet any cell debris.

9. Label a new microcentrifuge tube. Using a pipette, transfer up to
700 μL of supernatant (avoid contacting the pellet with the pipette tip) into the new microcentrifuge tube.

10. Add an equal volume of 70% ethanol to the lysate collected above
(100 μL of ethanol is added to every 100 μL of lysate). Vortex to mix.

#### Part II - binding DNA to column

1. Assemble a spin column with one of the provided collection tubes.

2. Apply 700 μL of the clarified lysate with ethanol onto the column
and centrifuge for 1 minute at 10,000 x g (~10,000 RPM). Discard the flowthrough and reassemble the spin column with the collection tube.

:::info
Ensure the entire lysate volume has passed through into the collection tube by inspecting the column. If the entire lysate volume has not passed, spin for an additional minute at 20,000 x g (~14,000 RPM).
:::

3. Repeat step 2 with the remaining volume of lysate mixture.

#### Part III - wash column

1. Apply 500 μL of Binding Buffer B to the column and centrifuge
for 1 minute at 10,000 x g (~10,000 RPM).

:::info
Ensure the entire Binding Buffer B has passed through into the collection tube by inspecting the column. If the entire wash volume has not passed, spin for an additional minute.
:::

2. Discard the flowthrough and reassemble the spin column with its
collection tube.

3. Apply 500 μL of Wash Solution A to the column and centrifuge for 1
minute at 10,000 x g (~10,000 RPM).

4. Discard the flowthrough and reassemble the spin column with its
collection tube.

5. Repeat steps 3 and 4.

6. Spin the column for 2 minutes at 20,000 x g (~14,000 RPM) in order
to thoroughly dry the resin. Discard the collection tube.

#### Part IV - Elute DNA

1. Place the column into a fresh 1.7 mL Elution tube provided with the kit.

2. Add 50 μL of Elution Buffer B to the column.

3. Centrifuge for 1 minute at 425 x g (~2,000 RPM), followed by a 1
minute spin at 20,000 x g (~14,000 RPM). If the entire volume has not been eluted, spin the column at 20,000 x g (~14,000 RPM) for 1 additional minute.

4. (Optional): An additional elution may be performed if desired by
repeating steps 2 and 3 using 50 μL of Elution Buffer. The total yield can be improved by an additional 20-30% when this second elution is performed.

The purified genomic DNA can be stored at 2-8°C for a few days. For longer term storage, -20°C is recommended.

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### Chelex Isolation for Plant Barcoding

- See the protocol on [DNA Barcoding 101](https://dnabarcoding101.org/lab/protocol-2.html#alternatec)

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### Student plant samples

Your number and your plant (e.g., Sample 7, coconut)

(Same as the Tuesday sign-in numbers, no sample 27)

## Results

1. Nanopore sequencing 16s Shoe-ome: [Samples 1-24](https://epi2me.nanoporetech.com/shared-report-449156?tokenv2=60d09295-e907-4136-9391-0e7bbd5ecdea)
2. Nanopore sequencing 16s Shoe-ome: [Samples 25-31](https://epi2me.nanoporetech.com/shared-report-449168?tokenv2=761e63e0-2464-42e9-9707-9f6018293e1c)
- Note that the samples and barcodes are as follows (samples numbered on March 4):
- Barcode 1: Sample 25
- Barcode 2: Sample 26
- Barcode 3: Sample 27
- Barcode 4: Sample 28
- Barcode 5: Sample 29
- Barcode 6: Sample 30
- Barcode 7: Sample 31
- All other barcodes shown are sequencing errors and have between 1-4 reads per sample shown
4. Plant DNA barcoding [Comming late next week]
- Note: No sample 27 so samples with higher numbers will need renaming
- Results available on DNA Subway"
- Tracking Number: **30-1002653735**
- Video tutorial: [Zoom recording](https://cshl-dnalc.zoom.us/rec/share/DzR8GcMgB3ynFIBOXnUtCtJzigAD6W1WeYarF5wRxqxDCrNlLaSyJgZhucIcrRc6.wQ5jWs132BPcAbpn)
- Extended DNA Subway walkthough: [DNALC Live](https://dnalc.cshl.edu/resources/dnalc-live/series-barcoding-bioinformatics.html)
- Extended DNA Subway packet: [Using DNA Subway to Analyze DNA Barcoding Sequences (Student Handout)](https://dnalc.cshl.edu/assets/files/dnalc-live/17192.using-dna-subway-to-analyze-sequence-barcodes-05182018-student.pdf)