GDSC-HyperTRIBE-pipeline

Pipeline for running HyperTRIBE to identify RNA editing sites

Table of Contents

• Introduction
• Pipeline
• Prerequisities
• MySQL
• Implementation
• Software
• References

Introduction

HyperTRIBE is a technique used for the identification of the targets of RNA binding proteins (RBP) in vivo. HyperTRIBE couples an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR and expresses the fusion protein in vivo. As the RBP-ADARcd (catalytic domain) fusion protein lacks the RNA recognition features of ADAR, the specificity of the RBP should determine the editing specificity of the fusion protein. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. HyperTRIBE identifies RNA editing sites (A to I change, where I is read as G) by comparing RNA sequence from transcriptome with wild type RNA (wtRNA) from the same background.

Pipeline

The major steps of this pipeline are as follows:

1. Trim and align sequence libraries to genome
2. Load alignment data to MySQL database
3. Find RNA A-->G edit sites against wtRNA-RNA sample

Prerequisites

This pipeline relies on a series of conda environments and a MySQL Instance through Dartmouth Dashboard. Should this instance fail, a new instance can be created following these instructions. Once an instance is generated, credentials can be obtained in the CLI section on Dartmouth Dashboard.

Anytime a new instance is generated, the MySQL credentials will need to be edited in the perl source code files in the CODE directory

The current credentials for the MySQL instance are as follows:

Host	Database	User	Password	Last Updated
dmseq-f11b-db.c.dartmouth.edu	dmseq	admin	gdscPass	April 26th, 2025

MySQL

To interactively use the MySQL instance, run the following command on your LOCAL termainl with the credentials above:

mysql -h dmseq-f11b-db.c.dartmouth.edu -u admin -p --ssl-mode=REQUIRED

CRUCIAL You need to ensure that mysql is allowing infile loading on both the client and server side. To check this: once inside mysql, run the following:

#-----Check
SHOW GLOBAL VARIABLES LIKE 'local_infile';

#-----If the output of this command returns the follwing, run the next line:
+---------------+-------+
| Variable_name | Value |
+---------------+-------+
| local_infile  | OFF   |
+---------------+-------+

#---- Run this is `local_infile` is off
SET GLOBAL local_infile = 1;

#---- Check again
+---------------+-------+
| Variable_name | Value |
+---------------+-------+
| local_infile  | ON    |
+---------------+-------+

Also ensure within your code folder, you have the mysql.cnf file with the following contents:

[client]
local_infile = 1
ssl-mode=REQUIRED

[mysqld]
local_infile = 1

Implementation

1. Clone the github repository

2. HyperTRIBE annotations can be found at the following path on Discovery /dartfs-hpc/rc/lab/G/GMBSR_bioinfo/genomic_references/hyperTRIBE_annotations/mm39. You can either symlink this path to your working directory and point to it in the config.yaml, or point directly to the source.

git clone https://github.com/Dartmouth-Data-Analytics-Core/GDSC-HyperTRIBE
cd GDSC-HyperTRIBE

3. Create a samples.csv file (comma-separated). This file is 4 columns. An example is below.

Sample_ID,fastq_1,fastq_2,replicate
DCD,data/211338_02_S14_R1_001.fastq.gz,data/211338_02_S14_R2_001.fastq.gz,DCD
Mut,data/211338_01_S13_R1_001.fastq.gz,data/211338_01_S13_R2_001.fastq.gz,Mut

4. Ensure all variables in config.yaml are edited to point to proper paths

5. Submit Snakemake job script

sbatch job.script.sh

6. Upon completion of Snakemake workflow, run the annotation script on the group of interest using CODE/hyoerTribe_Annotate.sh This script takes 4 arguments: path to xls results, any file name ending in .txt (for when data gets pivoted), output prefix, and path to annotation gtf. This script will create an Annotation directory where final results will live as a .tsv file.

bash CODE/hyperTribe_Annotate.sh \
    results/Mut.HyperTRIBE_results.xls \
    results/output.txt \
    MUT \
    Mus_musculus.GRCm39.114.gtf

Software

Tool	Version	Conda
Trimmomatic	0.39	align
bowtie2	2.5.4	align
star	2.7.11b	align
picard	3.4.0	rnaseq1 or align
samtools	1.15.1	samtools
perl	5.32.1	perl
perl-app-cpanminus	1.7048	perl
perl-dbi	1.643	perl
perl-dbd-mysql	4.050	perl
mysql	9.3.0	perl
bedtools	TBD	TBD

References

For more details please see:

HyperTRIBE ReadTheDocs

Xu, W., Rahman, R., Rosbash, M. Mechanistic Implications of Enhanced Editing by a HyperTRIBE RNA-binding protein. RNA 24, 173-182 (2018). doi:10.1261/rna.064691.117

McMahon, A.C., Rahman, R., Jin, H., Shen, J.L., Fieldsend, A., Luo, W., Rosbash, M. TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins. Cell 165, 742-753 (2016). doi: 10.1016/j.cell.2016.03.007.