# Nanopore Network NYC Workshop
*June 5-9, 2023*

----


*Attendees (please add your name once you have your HackMD account)**

1. Jason Williams, CSHL, [e-mail]
2. Jesus Alan Salas, Lehman College, [e-mail]
3. Xiomara Chianelli, EPCC, [e-mail]
4. Jeremy Seto, City Tech, [e-mail]
5. Andrew Lee, Northern VA Community College (Alexandria,VA), [e-mail]
6. Rodolfo da Silva Probst, Sci. Res. Iniatiative (University of Utah), [e-mail]
7. James Melton (Spelman College), [e-mail]
8. Miguel Urdaneta, University of Puerto Rico Rio Piedras (UPR-RP), [e-mail]
9. José L. Agosto Rivera,University of Puerto Rico Rio Piedras (UPR-RP), [e-mail])
10. Rudolf Bohm, Texas A&M University-Kingsville, [e-mail]
11. Gene Espinoza, Texas A&M University-Kingsville, [e-mail]
12. Luis E. Vázquez-Quiñones, Universidad Interamericana de Puerto Rico - Arecibo Campus, [e-mail]
13. Miguel A. Aguilar EPCC, [e-mail]
14. Doug Foster, [e-mail]
15. Cristina Andujar, [e-mail]
16. Trevor Duarte, El Paso Community College, [e-mail]
17. Anna Feitzinger, DNALC NYC [e-mail]
18. Maira Goytia, Spelman College, [e-mail]

## Manual update
~~~
sudo apt-key adv --fetch-keys https://cdn.oxfordnanoportal.com/apt/ont-repo.pub
sudo apt update
sudo apt install ont-mk1c-release
~~~

## Logistics

- **Location**: *DNALC NYC*, [62 Tillary st., Brooklyn, NY](https://goo.gl/maps/Z6J9hJW5tATdd7Cn6?coh=178572&entry=tt)
- Building entry is locked, guard will sign you in each morning, bring identification
- Staff have a badge can can assist you (we will circulate cell phone numbers)
- **Times**: Workshop will run 9:30 AM - 5:00 PM (M-Th), 9:30 AM - 12:00 PM Friday
- **Food**:
- AM/PM Coffee and snacks will be available
- Lunch is on-your-own M-Th/ Pizza lunch on Friday
- **What to bring**:
- Laptop: Please bring a laptop; we will do analyses on the cloud so no specific requirements. A small number of loan laptops should be available.
- DNA or samples, if you plan to sequence a particular non-pathogenic/non-human sample
- Reagents/kits (optional): We will have a variety of kits for DNA extraction, library prep, etc. flow cells, and devices. You may elect to bring your own, but this is not required. Refrigeration will be available.
- **Reimbursement and stipends**:
- Through [InnovATEBIO](https://innovatebio.org/) we are able to support travel and stipends for community college faculty.
- Other faculty will also be supported by NSF grants for [InnovATEBIO](https://innovatebio.org/acknowledgements) and the Nanopore [IUSE](https://www.nsf.gov/awardsearch/showAward?AWD_ID=2216349&HistoricalAwards=false) projects.
- Save all receipts for reimbursable expenseses. We will also have you complete reimbursement forms which will be processed after your successful completion of the workshop.

- **Other advanced prep**
- **Obtain a [HackMD](https://hackmd.io/login) account** so that you can edit this document.
- If you haven't done so, **ensure you have an account on the Oxford Nanopore website**: [register for an account](https://registration.nanoporetech.com/user/details/)
- We will be developing an eBook/website with learning materials, lab protocols, etc. **Visit the [Draft Nanopore eBook site](https://nanoporenetwork.github.io/ebook-website/Summer-2023-test-6/)** and consider how you would like to contribute. Most pages are placeholders currently and will explain how to contribute during the workshop.
- **Register for an [NSF ACCESS](https://identity.access-ci.org/new-user) account**; if you previously had an XSEDE account, the page at the link shows you how to connect your profile.

## Links and resources

**Websites**

- [Nanopore Network QUBES site](https://qubeshub.org/community/groups/nanoporenetwork)
- [Draft Nanopore eBook site](https://nanoporenetwork.github.io/ebook-website/Summer-2023-test-6/)
- [Oxford Nanopore Store](https://store.nanoporetech.com/us/)
- [Oxford Nanopore Community](https://nanoporetech.com/community)
- [Shared Google Drive](https://drive.google.com/drive/folders/18ESJWuRcprSelmFgTQxakl7u6pLhOAl9?usp=sharing)
- [Nanopore website protocol builder](https://community.nanoporetech.com/docs/plan/protocol_builder/aims?result=50448)

**Computing**

- [JetStream2](https://jetstream-cloud.org/)
- [NSF ACCESS Computing](https://access-ci.org/)
- [Nanopore EPI2ME workflows](https://epi2me.nanoporetech.com/workflows)
- [List of Nanopore sequencing tools](https://github.com/GoekeLab/awesome-nanopore)

**eBook authoring**

- [Mkdocs](https://mkdocs.readthedocs.io/en/stable/)
- [Nanopore network GitHub](https://github.com/nanoporenetwork/)
- [Material Mkdocs theme](https://squidfunk.github.io/mkdocs-material/)
- [AI generated documentation](https://get.scribehow.com/ai-scribe/)
- [Markdown cheatsheet](https://github.com/adam-p/markdown-here/wiki/Markdown-Cheatsheet)

## Agenda

### Monday

**AM**
- Introduction and goals for the week
- Overview of eBook [draft site here](https://nanoporenetwork.github.io/ebook-website/Summer-2023-test-6/)
- Break into Teams
- Experiment planning and coordination

**PM**
- Work on experiments
- Work together on some simple eBook contributions

### Tuesday
**AM**
- Presentation by Nanopore representatives
- Jonathan Pugh (Associate Director, Nanopore Education)
- Akelia Odumbo, Technical Services Scientist
- Team regroups and discussion
- Experimental lab work

**PM**

- Experimental lab work

### Wednesday
**AM**
- Experimental lab work
- Bioinformatics tutorials and labs
- Documentation and demo planning
- Wrap up lab work

**PM**
- Data management and analysis planning
- Bioinformatics tutorials and lab
- Bioinformatics; Introduction to Jupyter
- Discussion on shared teaching materials

### Thursday
**AM**
- experiment re/runs and bioinformatics
- eBook contributions

**PM**
- Demo filming
- Bioinformatics tutorials
- eBook contributions

### Friday
**AM**
- Workshop assessment/feedback (tentative)
- Fall FMN planning
- Group summary presentations
- Homework assignments
- Pizza lunch and certificates (12:00)

### Funding Acknowledgemnts

- Stipends and travel support made possible through [InnovATEBIO](https://innovatebio.org/acknowledgements)
- Additional support from NSF [IUSE](https://www.nsf.gov/awardsearch/showAward?AWD_ID=2216349&HistoricalAwards=false)

## Notes

## Monday

**Markdown tutorial**

|Col 1|Col 2|
|-----|-----|
|item 1|item 2

See - [Markdown cheatsheet](https://github.com/adam-p/markdown-here/wiki/Markdown-Cheatsheet)

---

### Experiment and team planning

|Name|Interest|Have sample|DNA Extraction|
|----|--------|-----------|--------------|
|Jason|DNA Barcoding (*rbcL*) using Nanopore|Yes, I have some plants |
| Jeremy |DNA metabarcoding (*fITS*, *pITS*, *rbcL*), Eukaryotic RNASeq, Metagenomics|No/Maybe|
|James |Soil metagenomics |Will obtain sample outside|
|Alan S |DNA Barcoding | No |
|Andrew|16s bacteria, whole genome, soil, interested in all! | No sample (yet)|
|Luis Vázquez|DNA Barcoding - Amplicon sequencing with José Agosto's team, RNA-seq & RNA-seq bioinformatics tools|José's sample|
|Doug Foster | DNA barcoding of sponge & symbionts|Yes Marine Sponge|
|Rodolfo Probst | WGS, metabarcoding|Yes, I have ant samples|[Promega Wizard® High Molecular Weight DNA Extraction (A2920)](https://www.promega.com/products/nucleic-acid-extraction/genomic-dna/high-molecular-weight-dna-extraction-kit/?catNum=A2920#protocols)
| Rudi B| Amplicon Sequencing with DNA barcoding|Not yet|
| Trevor Duarte |16s barcoding, ITS4, soil metagenomics | No|
| Miguel Aguilar| DNA barcoding for bacteria and phages| No|
|Anna Feitzinger |DNA barcoding/metabarcoding | Maybe|
Xiomara Chianelli |soil/water metagenomics or anything suitable for a CURE course|
| Jose L. Agosto Rivera| Amplicon Sequencing,PCR-based Barcoding|YES
| Cristina Andujar | DNA Barcoding | |
| Miguel Urdaneta| Amplicon Barcoding and Sequencing|Yes|
| Maira Goytia|Bacterial Transcriptomics, comparative genomics (WGS) |no
| | |

#### Teams

**Experiment(s)**: DNA Barcoding

- PCR amplicon based, eukaryotes, multiple loci
- **Kit**: Rapid(cDNA barcoding)
- **Members**: Anna
- Xiomara Chianelli
-

**Experiment(s)**: DNA Barcoding

- (PCR amplicon based, prokaryotes - 16s)
- **Kit**: Rapid
- **Members**: Miguel
- José
- Miguel Aguilar
- Luis Vázquez
- Cristina Andujar
- Gene Espinoza

**Experiment(s)**: Whole genomes
**DNA extraction?**: Yes

- Whole genome (Ants)
- **kit**: Ligation
- **Members**: Rodolfo, Jeremy, Alan, Rudi B
- [Wizard® High Molecular Weight DNA (A2920)](https://www.promega.com/-/media/files/resources/protocols/technical-manuals/500/wizard-hmw-dna-extraction-kit-protocol.pdf?rev=ee725be682a04f57a21438afa7debe51&sc_lang=en)

- Whole metagenome (soil)
- **kit**: Ligation
- **Members**: James, Andrew, Trevor, Rodolfo, Maira
- Ligation sequencing gDNA - Native Barcoding Kit 24 V14
(SQK-NBD114.24)

- Whole metagenome (Sponge) and symbionts
- **Kit**: 16s (bacterial symbionts/sponge microbiome)
- **Members**: Doug
- [Rapid sequencing amplicons - four primer PCR (SQK-PSK004 or SQK-PBK004)](https://community.nanoporetech.com/docs/prepare/library_prep_protocols/nested-pcr-protocol/v/ffp_9038_v108_revw_14aug2019)

**E-book interests**

##### Glossary
- Andrew
- RudiB
- Doug

##### Course-Based Research

- Trevor
- James

##### Sequencing Devices
- Rodolfo Probst

##### Laboratory Protocols
- Rodolfo Probst

:heavy_check_mark:
HotSHOT extraction

:heavy_check_mark:
DNA extraction (column-based Qiagen)

:heavy_check_mark:
Loading device (from library to sequencing)

:heavy_check_mark:
Qubit protocol

##### Sample Collection
- Rodolfo Probst (arthropods)

##### Curated Experiments (DNA Barcoding)
- Rodolfo Probst

##### Data Organization and Management
- Rodolfo Probst

##### Helpful Videos
- Carolina

Helpful Nanopore videos and videos for lab techniques for students. Example of videos for students: how to pipette, how to perform dilutions. Would also like to add links such as DNA Barcoding 101 which gives the different options for DNA isolation of different sample types, and femtomolar conversion link for conversion to fmol from ng/μl, as some examples.

##### PCR amplicon based, prokaryotes - 16s, Instruction Material
- Cristina Andújar
- José Agosto Rivera
- Miguel Urdaneta Colón
- Luis Vázquuez Quiñones

##### Guide To Creating Your Nanopore Account (english y español)
- Miguel Aguilar

##### Videos Recorded during Nanopore Network Workshop NYC, June 5-9, 2023

## Hackpad team invite
[invite link](https://hackmd.io/join/rkXPo8Jwh)

## Bioinformatics

[FastQC](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)

### Jupyter Instances

Place your name next to an IP address. Two people per IP teams if possible:

- (5/17): http://149.165.169.164:8001, users: Carolina
-
- (6/17): http://149.165.172.32:8001, users:Cristina
- (7/17): http://149.165.172.41:8001, users: Andrew, Gene
- (12/17): http://149.165.169.208:8001, users: jeremy
- (14/17): http://149.165.172.149:8001, users:RudiB
- (17/17): http://149.165.170.137:8001, users:James, Rodolfo
- (8/17): http://149.165.172.107:8001, users:Trevor
- (13/17): http://149.165.175.23:8001, users: Doug

- (16/17): http://149.165.152.86:8001, users: Jason
- (r1/7): http://149.165.173.18:8001, users: Miguel
- (r3/7): http://149.165.168.189:8001, users:Anna
- (r-5/7): http://149.165.171.109:8001, users:Miguel U
-
- (r/7/7): http://149.165.170.139:8001, users: Luis

**Credentials**

**Notebook**

*For viewing*
[FastQC notebook](https://raw.githubusercontent.com/JasonJWilliamsNY/genome_camp_2021/main/genome_camp_2022/notebooks/chamecrista_FastQC-notebook_jupyter.ipynb)

wget https://raw.githubusercontent.com/JasonJWilliamsNY/genome_camp_2021/main/genome_camp_2022/notebooks/chamecrista_FastQC-notebook_jupyter.ipynb

FLOW CELL comparision article
* Article: Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction. (2023)
Sanderson et al., Microbial Genomics 2023;9:000910 DOI 10.1099/mgen.0.000910 ([PDF](https://www.microbiologyresearch.org/docserver/fulltext/mgen/9/1/mgen000910.pdf?expires=1686236468&id=id&accname=guest&checksum=EEBD8BBBAAC09C9D4F233A8BCE32F49A))

**Pizza**
[Norms pizza](https://normspizza.com/welcome#menu-home)

*Any requests*

Margherita

Salad or gluten-free

Vodka Sauce looks interesting

Salad

cheese pie

**Last Minute Requests**

* Review of minION wash procedure- Flow cell model specific
* Cost of DNA bar coding for species ID: Sangers via Genewiz vs minION
* As a beginner, what parameters should you monitor to tell whether you have enough data (& can thus stop to save pores )

- Future Directions and Possible Connections

- Coordinating ordering for bulk discounting

- Cost accounting common experiments

- Step by step for moving data from wherever the sequencer stored it to wherever it needs to be for Jupyter. Specifically how did you create the project folder with a particular user name that is in the chamecriste FastQC tutorial.

- Protocols for testing water for presence of species (environmental DNA)

### Future Directions and Possible Connections

Name, and next projects!

Andrew: creating stripped down 16S skin microbiome protocol with basic bioinformatics analysis via Epi2me (demonstration running in summer, fall; unclear when deployed to classroom).

James: isolating and sequencing phage genomes from soil

Rudi B: 16s for Microbiology CURE *Plegechelia* plant genome assembly, pesticide resitance amplicon surveillence, mutant Fly Genome

Jose, Miguel, Cristina, Luis -
1_Prepare protocols and materials to implement amplicon sequencing (16S) into an experimental section of the genetics lab.
2_Continue learning whole-genome assembly and get ready to implement it in the genetics lab.

Anna (***La Jefa***): Optimizing Nanopore for DNA barcoding

Jeremy: MLST using barcode kits, Figuring out the convoluted eukaryotic RNAseq

Carolina: 16S and environmenal sequencing

Rodolfo: Metazoarian barcodes (maybe up to mitogenomes), ant-plant symbioses, ant genomes, phylogeography, potentially transcriptomics, species discovery with custom-indexing.

Trevor,Miguel: 16s DNA barcoding from soil extracts for 16 week 1st semester undergraduate general biology lab section.

Doug: cheap DNA barcoding for species ID

[Qubes Hub](https://qubeshub.org/community/groups/nanoporenetwork)

Video

### Data request

- Do you need us to send data?

Trevor Duarte, James Melton. Yes, please send me the data that we ran from our soil extracts. Barcodes 5-8
Carolina.

:::success
Data for the run above can be found in Rodolfo's Drive. Link [here](https://drive.google.com/drive/folders/1OuPzKzXPJxh_8vewrs4iix0RenjSGzJr)
:::

- What do you need help with?

Rodolfo: help with WGS analyses, Hi-C pipeline, assembly process for genomics and metagenomics.