Quantify IR

Either FASTQ files or BAM files can be used by IRFinder to quantify IR.

FastQ Mode (default)

This mode provides an end-to-end IR measurement. It aligns FastQ reads against STAR reference while quantifies IR on the fly. This mode generates both an alignment file (unsorted BAM) and IR analysis results.

$ bin/IRFinder -r REF/Human-hg19-release75 -d SRR900288_irfinder \    
  SRR900288_1.fastq SRR900288_2.fastq

In case you want to use IRFinder to carry out the adaptor trimming and alignment, we recommend unzipping .fastq.gz files first.
An example for aligning paired-end RNA-seq with adaptor trimming (two adaptors separated by space):

$ bin/IRFinder -r REF/Human-hg19-release75 -d SRR900288_irfinder \    
  -a AGATCGGAAG AGATCGGAAG \    
  SRR900288_1.fastq SRR900288_2.fastq

An example for aligning Single-end RNA-seq with adaptor trimming:

$ bin/IRFinder -r REF/Human-hg19-release75 -d SRR900288_irfinder \    
  -a AGATCGGAAG \    
  SRR900288.fastq

Please note, under FastQ mode, IRFinder uses the following STAR arguments to align the RNASeq reads while calculating IR:

--genomeLoad
--runThreadN
--genomeDir
--outFilterMultimapNmax 1
--outSAMstrandField intronMotif
--outFileNamePrefix
--outSAMunmapped None
--outSAMtype BAM Unsorted
--outStd BAM_Unsorted
--readFilesIn
--readFilesCommand

It is possible to parse user-defined additional STAR arguments by -y option under FastQ mode. For example, the following command tells STAR alignment to only consider reads whose mismatch ratio is below 0.2, so that IRFinder will only quantify IR on those reads:

$ bin/IRFinder -r REF/Human-hg19-release75 -d SRR900288_irfinder \    
  -a AGATCGGAAG AGATCGGAAG \    
  -y "--outFilterMismatchNoverLmax 0.2" \
  SRR900288_1.fastq SRR900288_2.fastq

If you encountered this issue described in the Troubleshoot session, please first try the solution provided there.

BAM Mode (-m BAM)

We highly recommend using BAM mode if users have already aligned RNASeq reads by STAR before running IRFinder. In this mode, IRFinder directly quantifies IR without re-aligning the reads again. This benefits the analysis consistency that all the results, including those from IRFinder and other RNASeq-related analysis, derived from the same alignment. This is because the default STAR call by IRFinder might be different from the STAR call of the original alignment, as aforementioned.

The following command will implement IR quantification on a BAM file and save the results in the new SRR900288_irfinder folder, defined by -d option.

#WARNING: for paired-end BAM input, it MUST be sorted by name (a.k.a unsorted).  

$ bin/IRFinder -m BAM -r REF/Human-hg19-release75 -d SRR900288_irfinder \    
  Unsorted.bam

If you found no read mapped after the BAM-mode analysis as described in the Troubleshoot session, please first try the solution provided there.