Run EpiNano on single gene transcripts

[Guoming-Gao]

We are interested in using the EpiNano developed in your group to analyze m6A modifications via RNA direct sequencing. The aim is to confirm the m6A modifications on RNAs transcribed from a single gene.

I followed the configurations and steps in the paper, EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing, using a reference fasta file containing a single gene rather than a genome or transcriptome.

I run:

python ~/EpiNano/Epinano_Variants.py -n 6 -R ~/AS/m6a_construct_RNA.fa -b ~/AS/fastq_runid_ee1fefbf2a6d42535fdf1461936a093d220dca7d_0.bam -s /home/gmgao/jvarkit/dist/sam2tsv.jar --type g

The following error came up when using Epinano_Variants.py:

/home/gmgao/AS/m6a_construct_RNA.fa.fai needs to be created with samtools faidx

I then used samtools faidx m6a_construct_RNA.fa to create the fai file and run Epinano_Variants.py again. However, a second error came up which I cannot solve:

m6a_construct_RNA.fa.dict needs to be created using picard.jar CreateSequenceDictionary

Note that I have installed all required software and packages in the specified versions using conda.

Are there any specific preparation steps of the reference fasta file that should be done prior to being fed into EpiNano?

[Guoming-Gao]

I have not tested it using the test data because I cannot find a way to use the test data on the starting step, which is Epinano_Variants.py. Can anyone help? Thanks a lot!

[Huanle]

Hi @Guoming-Gao ,

It reminds you to create a dictionary file for your reference. Please have a look at the wiki . java -jar picard.jar CreateSequenceDictionary R=transcriptome.fasta

There are test data in the test_data folder.

[Guoming-Gao]

Thank you so much for pointing us to the wiki! That's very helpful! @Huanle

Let me try running it today and see if the problem could be solved.