# BYOE Nanopore Drop In Workshop

*Hosted by [InnovATEBIO](https://innovatebio.org/) and the [DNA Learning Center](https://dnalc.cshl.edu/)*

## Day 1 - DNA and Library Prep:

10:00 -10:30 Introductions and Survey to technology/ goals for workshop
10:30 -12:00 Extraction of DNA (contingent on organism)
12:00-1:00 Lunch
1:00- 2:00 Quality control of DNA
2:00 - 4:00 Library prep
4:00 - 5:00 Quality control of library prep

## Day 2 - Sequence Runs and Bioinformatics

10:00 -12:00 Load Sequencers, more Nanopore technology background
12:00 -1:00 Lunch
1:00 - 2:00 File organization, management, and data transfer
2:00 - 3:00 Background on bioinformatics tools; basecalling (guppy?),
sequence quality (fastqc, or other?), alignments/ assemblies
4:00 - 5:00 Installation and introduction to Jupyter

## Day 3 - Additional Bioinformatics

10:00 - 12:00 Working through Jupyter notebooks on QC and filtering
12:00 - 1:00 Lunch
2:00 - 3:00 Survey of Nanopore tools and installation
4:00 Wrap up and next steps planning

## DNA Extraction Protocols

Wizard® HMW DNA Extraction Kit
https://www.promega.com/-/media/files/resources/protocols/technical-manuals/500/wizard-hmw-dna-extraction-kit-protocol.pdf?rev=6e15629ebf0040db89ef439f720f67d9&sc_lang=en

Nanobind Plant Nuclei Big DNA Kit Handbook
https://15a13b02-7dac-4315-baa5-b3ced1ea969d.filesusr.com/ugd/5518db_fcf7a543b1c54b3da181f96176d1d2df.pdf?index=true

Nanobind CBB Big DNA Kit Handbook (Cells, Blood, Bacteria)
https://15a13b02-7dac-4315-baa5-b3ced1ea969d.filesusr.com/ugd/5518db_3d2c66a5e3e74c24b313966fcf3fecdf.pdf?index=true

Nanobind Tissue Big DNA Kit Handbook
https://15a13b02-7dac-4315-baa5-b3ced1ea969d.filesusr.com/ugd/5518db_f106affdb20c458bab616598484b0e95.pdf?index=true

Nuclei Isolation – LN2 Plant Tissue Protocol
https://www.circulomics.com/_files/ugd/5518db_ae5fcdbc8c034085b191b80c6e5572dc.pdf?index=true

## Qubit Protocol

Qubit dsDNA HS Assay
https://tools.thermofisher.com/content/sfs/manuals/Qubit_dsDNA_HS_Assay_UG.pdf

## Library Prep Protocols

Rapid Sequencing Kit
https://community.nanoporetech.com/docs/prepare/library_prep_protocols/rapid-barcoding-sequencing-sqk-rbk004/v/rbk_9054_v2_revab_14aug2019

https://community.nanoporetech.com/docs/prepare/library_prep_protocols/rapid-barcoding-sequencing-sqk-rbk004/v/rbk_9054_v2_revab_14aug2019

Rapid sequencing amplicons - 16S barcoding
https://community.nanoporetech.com/docs/prepare/library_prep_protocols/16S-barcoding-sequencing/v/rab_9053_v1_revp_14aug2019

Ligation Sequencing Kit
https://store.nanoporetech.com/us/ligation-sequencing-kit-112.html

## Software tools
**Nanopore**
- Poretools: Toolkit for working with nanopore sequencing data from Oxford Nanopore - https://poretools.readthedocs.io/en/latest/
- Nanopolot: Plotting tool for long read sequencing data and alignments - https://github.com/wdecoster/NanoPlot
- NanoFilt: Filtering on quality and/or read length, and optional trimming after passing filters - https://github.com/wdecoster/nanofilt
- NanoStat: Calculate various statistics from a long read sequencing dataset - https://github.com/wdecoster/nanostat
- Nanoreviser: https://github.com/pkubioinformatics/NanoReviser
- NECAT Nanopore data assembler: https://github.com/xiaochuanle/NECAT

**Alignment**
- Graphmap: https://github.com/isovic/graphmap
- Graphmap (conda): https://bioconda.github.io/recipes/graphmap/README.html?highlight=graphmap#package-package%20'graphmap'
- Graphaligner (conda): https://bioconda.github.io/recipes/graphaligner/README.html?highlight=graphaligner#package-package%20'graphaligner'

**Genome assembly**
- Redbean: https://github.com/ruanjue/wtdbg2
- Flye: https://bioconda.github.io/recipes/flye/README.html?highlight=flye#package-package%20'flye'
- CANU (conda): https://bioconda.github.io/recipes/canu/README.html?highlight=canu#package-package%20'canu'

- MitoVGP: VGP Mitochondrial assembly pipeline https://github.com/gf777/mitoVGP

**Assembly Quality**
- QUAST: https://github.com/ablab/quast
- BUSCO: gene content of near-universal single-copy orthologs - https://busco.ezlab.org/
- BUSCO (conda): https://bioconda.github.io/recipes/busco/README.html?highlight=busco#package-package%20'busco'
- SQUAT: https://github.com/luke831215/SQUAT
- GenomeQC: https://github.com/HuffordLab/GenomeQC

## Data management

- Cyberduck: [Download](https://cyberduck.io/)
- How to configure Cyberduck: [CyVerse learning center](https://cyverse-learning-materials.github.io/cyverse_mooc/step5/)

## Online accounts

Please make sure you have an account on the following platforms:

- CyVerse [https://cyverse.org/](https://cyverse.org/)
- Self guided [tutorial](https://cyverse-learning-materials.github.io/cyverse_mooc/)
- Cyverse FastQC example path: `/iplant/home/shared/cyverse_training/classrooms/dnalc_genome_camp/spolyrhiza_08_2021/spolyrhiza_08252021/reads_for_assembly/guppy_gpu_fastq_files/concat_fastq`
- Nanopore [https://nanoporetech.com/community](https://nanoporetech.com/community)

## Protocols