HAMRbox: A user-friendly adaptation of HAMR

Overview

• HAMRbox is a bundle of selected tools that capture different parts of the pipeline tailored for the High Throughput Annotation of Modified Ribonucleotides, abbreviated HAMR, developed by Paul Ryvkin et al. HAMRbox aims to make the original method more accessible by automating the tedious pre-processing steps, allowing users to analyze RNA-seq data at an experiment scale.
• HAMRbox is high-throughput and performs RNA-modification analysis at a bioproject scale. HAMRbox performs constitutive trimming of acquired reads using Trim-Galore, and makes use of STAR as the aligning tool which reduces runtime with a rivaling robustness compared to TopHat2 as suggested in the original pipeline.

Command Line Arguments and Description

Command	Description
Required
-o	<project directory> where you want your entire hamr project to be
-t	<SRA accession list.txt> or <folder of raw fastq files> a txt file of all srr accession code to your desired reads or a path containing them
-c	<filenames for each fastq.csv> a csv file that corresponds each srr code (or name of fastq file) to your desired nomenclature for each read
-g	<reference genome.fa> a fasta file of the genome of the model organism
-i	<reference genome annotation.gff3> a gff3 file of the genome of the model organism, note we require gff3 instead of gtf
-l	<read length> an integer, the read length of this sequencing experiment, if non-unanimous use the shortest length
-s	<genome size in bp> an integer, the number of base pairs of the genome of this model organism
-e	<genome annotation generator code> see below for abbreviation code, one code per organism/cultivar
Optional
-a	[use Tophat2 instead of STAR] default uses STAR
-b	[Tophat2 library choice: fr-unstranded, fr-firststrand, fr-secondstrand] default=fr-firststrand
-f	[filter] default=filter_SAM_number_hits.pl
-Q	[HAMR: minimum qualuty score] default=30
-C	[HAMR: minimum coverage] default=50
-E	[HAMR: sequencing error] default=0.01
-P	[HAMR: maximum p-value] default=1
-F	[HAMR: maximum FDR] default=0.05
-m	[HAMR model] default=euk_trna_mods.Rdata
-n	[number of threads] default=4
-h	[help message]

Annotation Generator Code

Abbreviation Code	Organism
AT	Arabidopsis thaliana
BD	Brachypodium distachyon
ZM	Zea mays
OSJ	Oryza sativa japonica
OSI	Oryza sativa indica
OSIR64	Oryza sativa IR64

Running HAMRbox (demo)

Pull docker image from this git

docker pull harrlol/hamrbox

Download genome fastq file for Arabidopsis thaliana

wget -qO- https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-57/fasta/arabidopsis_thaliana/dna/Arabidopsis_thaliana.TAIR10.dna.toplevel.fa.gz | tar xvz 

Download annotation gff3 file for Arabidopsis thaliana

wget -qO- https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-57/gff3/arabidopsis_thaliana/Arabidopsis_thaliana.TAIR10.57.gff3.gz | tar xvz 

Run HAMRbox

docker run \
  --rm \
  -v $(pwd):/working-dir \
  -o /working-dir \
  harrlol/hamrbox \
  -t ~/demo/PRJNA596803_list.txt \
  -c ~/demo/PRJNA596803_filenames.csv \
  -g ~/path/to/genome.fasta \
  -i ~/path/to/genomeannotation.gff3 \
  -l 50 \
  -s 135000000 \
  -e AT