HAMR takes in a coordinate-sorted BAM file with mapped reads. Reads should be mapped with m>0 mismatches: patterns of mismatches will be used to detect and predict identity of modifications. HAMR outputs predicted modification sites including identity of the modifications.

NOTE: Spliced alignments (with N in CIGAR string) in BAM will be ignored.
Alignments with insertion/deletions will be ignored. 
Only continuous, un-interrupted read alignments will be used.
Any spliced alignments should be resolved (e.g., by splitting) before
running HAMR, e.g., using GATK
java -jar GenomeAnalysisTK.jar -T SplitNCigarReads -R <genome.fa> -I <input.bam> -o <input.splitN.bam> -U ALLOW_N_CIGAR_READS

Installing HAMR: 
HAMR includes pre-compiled binary programs.
If neccessary, to re-compile use
make clean
make

Operating instructions:

Usage:
python hamr.py [align.bam] [genome.fa] [prediction_model_file] [output_dir] [output_prefix] [min_read_qual] [min_read_coverage] [seq_error_rate] [hypothesis] [max_p] [max_fdr] [min_ref_percent] [OPTIONS]

Examples:
# download reference genome (e.g., hg19)
wget http://tesla.pcbi.upenn.edu/hamr/genomes/hg19_all_chr.fas

# run HAMR in genome-wide mode
python hamr.py trial.human.bam  genomes/hg19_all_chr.fas models/euk_trna_mods.Rdata HAMRtest human 30 10 0.05 H4 0.01 0.05 0.05

# run HAMR in target mode (BED-restricted)
python hamr.py trial.human.bam  genomes/hg19_all_chr.fas models/euk_trna_mods.Rdata HAMRtest human_region 30 10 0.05 H4 0.01 0.05 0.05 --target_bed region.human.bed

	
NOTE: Python with version v2.7.x is preferred. Rscript and samtools are required for running HAMR (make sure they are in searchable path).


REQUIRED arguments:
	<align.bam>
		input BAM file
	<genome.fa>
		reference genome FASTA file (should be the same as the genome file used in mapping (input BAM). NOTE: remember to index the reference genome using samtools faidx before running HAMR.
	<min_read_qual>
		the minimum base calling quality score. All low-quality bases will removed from analysis
	<min_read_cov>
		the minimum read coverage of a genomic position for it to be analyzed
	<seq_error_rate>
		The expected percentage of mismatches based solely on sequencing error
	<hypothesis>
		The hypothesis to be tested, either "H1" or "H4" (see HAMR
paper. H4 is recommended).
	<max_p>
		the maximum p-value cutoff. All sites with P-value><max_p>
will be filtered out.
	<max_fdr>
		the maximum FDR cutoff
	<refpercent>
		the minimum percentage of reads that must match the reference nucleotide. All sites with reference read nucleotide proportion < <refpercent> will be filtered out
	<output_dir>
		directory for the HAMR output
         
        <output_prefix>
		prefix for HAMR output files
 

OPTIONS:
	--target_bed <targets.bed>
		BED file with regions of interest for HAMR analysis (this replaces the default genome-wide HAMR mode and restricts HAMR analysis to specific genomic regions listed in BED)
	--paired_ends, -pe
		indicates paired-end sequencing was used 
	--filter_ends,-fe
		excludes the first and last positions in the read from the analysis



Copyright:
	Copyright (c) 2013-2016 University of Pennsylvania

	Permission is hereby granted, free of charge, to any person obtaining a
	copy of this software and associated documentation files (the "Software"),
	to deal in the Software without restriction, including without limitation
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	and/or sell copies of the Software, and to permit persons to whom the
	Software is furnished to do so, subject to the following conditions:

	The above copyright notice and this permission notice shall be included in
	all copies or substantial portions of the Software.

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