IRFinder Output

Files in the Ouput Folder

• IRFinder-IR-nondir.txt
  The main output file listing metrics for all measured introns. This is always an output no matter the input data is directional or non-directional RNA-Seq.
• IRFinder-IR-dir.txt
  The equivalent main output file, only generated for directional RNA-Seq, with analysis completed using strand-specific detail. There is no need to tell IRFinder if your data is non-directional / first-strand / second-strand - it decides automatically and outputs the relevant analysis.
• IRFinder-ChrCoverage.txt
  Number of reads mapped to each of the chromosomes. The last three columns represent total number of reads, number of reads on the positive strand and number of reads on the negative strand, respectivly.
• IRFinder-JuncCount.txt
  Number of reads mapped to splice junctions, in IRFinder output format. The last three columns represent total number of reads, number of reads on the positive strand and number of reads on the negative strand, respectivly.
• IRFinder-ROI.txt
  Number of reads mapped to the regions of interest (ROIs), if ROIs have been provided during IRFinder reference preparation. The last three columns represent total number of reads, number of reads on the positive strand and number of reads on the negative strand, respectivly.
• IRFinder-SpansPoint.txt
  Number of reads overhang splice junctions. The last three columns represent total number of reads, number of reads on the positive strand and number of reads on the negative strand, respectivly.
• irfinder.stderr
  Standard error during IRFinder execution.
• irfinder.stdout
  Standard output during IRFinder execution.
• Log.final.out
  STAR alignment final output log file. See STAR manual for details. FASTQ mode only.
• Log.out
  STAR execution log file. See STAR manual for details. FASTQ mode only.
• Log.progress.out
  STAR execution progress log file. See STAR manual for details. FASTQ mode only.
• Log.std.out
  STAR execution standard output file. See STAR manual for details. FASTQ mode only.
• SJ.out.tab
  Number of reads mapped to splice junctions, in STAR junction output format. See STAR manual for details. FASTQ mode only.
• trim.log
  Trimming information implemented by IRFinder. FASTQ mode only when -a is not set to none.
• Unsorted.bam
  BAM output for STAR alignment, sorted by read names. FASTQ mode only.
• WARNINGS
  This file records suspicious issues when quantifying IR using your input file.

Column Explanation for IRFinder-IR-dir.txt and IRFinder-IR-nondir.txt

Column1: Chr
Chromosome of the intron.

Column2: Start
0-indexed start position of the intron.

Column3: End
0-indexed coordinate indicating the end of an intronic region. Please note, the range of an intronic region is half-open, e.g.[start,end), follows the same rule of a BED format.

Column4: Gene Symbol/Ensembl ID/Static Warning
In which gene the intron locates. Static warning indicates if an annotated intron is overlapped with other features such as an alternative exon.

Column5: Null
This column is not used. Retained to maintain the first 6 columns as BED format.

Column6: Strand
Strand of the intron.

Column7: ExcludedBases
This is the number of bases within the intronic region that have been excluded from the calculation of intronic coverage because of overlapping features or mapping issues.

Column8: Coverage
Ratio of bases with mapped reads.

Column9: IntronDepth
Depth is the number of reads that map over a given bp. IntronDepth is the median depth of the intronic region without the excluded regions. It is used to calculate the IRratio. Excluded regions comprise ExclBases and bases with the top and bottom 30% of intronic depth.

Column10/11/12: Intron25/50/75Percentile
25th/50th/75th percentile of intronic depth.

Column13: ExonToIntronReadsLeft
This is the number of reads that overlap the 5' flanking exon and the intron.

Column14: ExonToIntronReadsRight
This is the number of reads that overlap the 3' flanking exon and the intron.

Column15/16: IntronDepthFirst/Last50bp
This is the depth of intron reads in the first and last 50bp of the intron.

Column17: SpliceLeft
This is the number of reads that map the 5' flanking exon surrounding the intron and to another exon within the same gene.

Column18: SpliceRight
This is the number of reads that map the 3' flanking exon surrounding the intron and to another exon within the same gene.

Column19: SpliceExact
This is the number of reads that map across the 3' and 5' flanking exons.

Column20: IRratio
When IntronDepth >=1:
IRratio = IntronDepth / (max(SplicesRight , SplicesLeft) + IntronDepth)
When IntronDepth < 1:
IRratio = Coverage / (max(SplicesRight , SplicesLeft) + Coverage)

Column21: Warnings
The warining tag is generated from the values in the previous columns, and the cutoff is hard-coded. There are five possible warning tags, namely LowCover, LowSplicing, MinorIsoform, NonUniformIntronCover and -. Specifically,

LowCover: the overall sequencing depth for an event is low. 
Judgement: (Column19 + Column9) < 10   

LowSplicing: not enough reads supporting the correct splicing.
Judgement: Column19 < 4
 
MinorIsoform: the event is not the major/most common splicing outcome among all transcripts of this gene.
Judgement: Column19 * 1.33333333 < max(Column17, Column18)
   
NonUniformIntronCover: a "dramatic" drop of read depth in the intronic region compared to read depth of the flanking exonic regions around splice junctions.
Judgement: max(Column13, Column14) > 2 + Column9 && max(Column13, Column14) > Column9 * 1.5

-: no warning
Judgement: no other warning tag is generated.