Editing Nanopore RNA Sequencing Protocol (section)

===Software===

* '''Biopython'''. Requires a specific version for ''EpiNano''.
* [https://docs.conda.io/en/latest/ Conda].
* '''Dask'''. Requires a specific version for ''EpiNano''.
* [https://community.nanoporetech.com/protocols/Guppy-protocol/ Guppy 6.4.2† for Linux].
* '''h5py'''. Requires a specific version for ''EpiNano''.
* [https://java.com/en/download/ Java Runtime Version 8+].
* [https://github.com/lh3/minimap2 Minimap2].
* '''Nanopolish'''. Requires a specific version for ''EpiNano''.
* '''Numpy'''. Requires a specific version for ''EpiNano''.
* '''Pandas'''. Requires a specific version for ''EpiNano''. Installed automatically with ''m6ANet''.
* [https://github.com/broadinstitute/picard/releases/tag/2.27.4 Picard].
* [https://pypi.org/project/pip/ Pip]. Python package installer. Installed automatically with ''m6ANet''.
* [https://cran.r-project.org/src/base/R-4/ R 4.1.2 (Bird Hippie)].
* [https://www.rstudio.com/products/rstudio/download/#download RStudio Desktop 2021.09.2+382].
* [http://www.htslib.org/ Samtools].
* '''Scikit-learn'''. Requires specific version for ''EpiNano''. Most recent version used for ''m6ANet''.
* [https://pytorch.org/ Torch 1.6.0].
''†Guppy 6.4.2 is used here since it is the most recent algorithm version. EpiNano-SVM’s pretrained models were performed with Guppy 3.1.5, but that is [https://community.nanoporetech.com/posts/proposed-changes-to-the-fa no longer compatible with newly acquired .fast5 data]. Additionally, Guppy 3.1.5 does not have GPU support, which makes processing data take much longer. I will keep the EpiNano-SVM code in here, as well as how to install Guppy 3.1.5, but you should use Guppy 6.4.2 and m6ANet instead.''

''Linux Base''

* '''Bash 3.2+'''.
* '''Cmake'''.
* '''Gfortran'''.
* '''Libidn11''' 32-bit libraries (if using old ''Guppy'' versions on a 64-bit machine outside of a 32-bit container).
* '''pyfaid'''.

''R Packages''

* [https://www.rdocumentation.org/packages/car/versions/3.1-1 car]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/dplyr/versions/1.0.10 dplyr]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/forcats/versions/0.5.2 forcats]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/ggplot2/versions/3.4.0 ggplot2].
* [https://www.rdocumentation.org/packages/ggrepel/versions/0.9.2 ggrepel].
* [https://www.rdocumentation.org/packages/gridExtra/versions/2.3 gridExtra].
* [https://www.rdocumentation.org/packages/optparse/versions/1.7.3 optparse]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/outliers/versions/0.15 outliers]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/purrr/versions/0.3.5 purrr]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/readr/versions/2.1.3 readr]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/reshape2/versions/1.4.4 reshape2]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/stringr/versions/1.5.0 stringr]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/tibble/versions/3.1.8 tibble]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/tidyr/versions/1.2.1 tidyr]. Required for ''EpiNano''.
* [https://www.rdocumentation.org/packages/tidyverse/versions/1.3.2 tidyverse].

Note that we will install all of these below if you do not have them. For those specified to require a specific version for ''EpiNano'', we will cover those in the section dedicated to ''EpiNano'' below. If you are detecting m6A modifications using ''m6ANet'', you don’t need to worry about the ''EpiNano'' requirements. The ''m6ANet'' requirements should be installed automatically.

====Getting Reference Transcriptome====

{{warning|A reference '''transcriptome''' is required for using ''m6ANet'', but you may choose to use the genome if you are using ''EpiNano''.}}

We will also need to download an appropriate reference transcriptome to map our reads to. I prefer to use the <code>GRCh38.p14</code> version, but you can use any you like. Just keep in mind that different reference assemblies may refer to transcripts or chromosomes using different IDs, which will change what you need to enter in the <code>${CHR}</code> (genome reference only) definition at the start of the protocol. Also, you can choose your own directory to place the reference transcriptome into, but just know that this path should also be updated in the <code>${REF}</code> shortcut in the Defining Files section above.

{{note|If the <code>wget</code> command is not available already on your system, you can install it with <code>sudo apt-get install wget</code>.}}

<code>Bash</code>
<syntaxhighlight lang="Bash">
cd "/home/$USER/Research/Ref"
wget "https://ftp.ncbi.nlm.nih.gov/genomes/all/annotation_releases/9606/110/GCF_000001405.40_GRCh38.p14/GCF_000001405.40_GRCh38.p14_rna.fna.gz"
gzip -d GCF_000001405.40_GRCh38.p14_rna.fna.gz
mv GCF_000001405.40_GRCh38.p14_rna.fna GRCh38.p14.rna.fna
</syntaxhighlight>

Note that here, I also renamed the transcriptome after extracting it for simplicity and ease of reference. A full list of all available genomes and transcriptomes can be accessed using the [https://ftp.ncbi.nlm.nih.gov/genomes/ NIH FTP], including older or more recent versions if you wish. You may also consider getting a reference genome / transcriptome from [https://useast.ensembl.org/index.html Ensembl] or [https://www.gencodegenes.org Gencode].