How to find your RNA-Seq data is unstranded or stranded

If you cannot figure out the strandness of your RNA-Seq data by BLAT or tags of SAM file -- there are couple of very nice documents on the web --  here is an alternative with IGV and I built a script based on that idea.

With the IGV

1. Generate a BAM file (by mapping to genome with your preferred aligner)
2. Generate an index by SAMTools.
3. Import the BAM file in the IGV.
5. Right click -> Color alignemnt by -> first-in-pair strand

If your data is stranded, you can see some dominant color in the area.

Stranded	Unstranded

With a script

Based on this, I built the following script for detecting strandness. 
It will check reads on the GAPDH or first 3 IDs of the GTF file and report the ratios of the major strand. The strandness will be determined if more than 90% of majority reads share the same strand. 

Save it as 'strandness.sh' and add executable permission. ('chmod a+x strandness.sh')
It will check if it doesn't have a index file or it mismatched organism. 
You need to install samtools first to use this script.

Usage) 

 a) Checking human (hg19)

 strandness.sh test.bam 

 or 

 strandness.sh test.bam human 

 b) Checking mouse (mm9)

 strandness.sh test.bam mouse 

 c) Checking with GTF file 

 strandness.sh test.bam test.gtf 


Output) 

 Checking standness script v0.01
 Input file: unstrand.bam
 GTF file  : ../goat_genes.gtf

 85.57% of reads (4409/5152) are pos-strand in NC_005044.2
 48.94% of reads (557854/1139785) are neg-strand in NC_022293.1
 45.06% of reads (846306/1877961) are neg-strand in NC_022294.1
 The data looks like un-stranded.

Script)

echo -e "Checking standness script v0.01\n"   #Default test region is Human GAPDH  REGION="chr12:6643571-6647541"  if [ -z "$1" ]  then     echo -e "\nNo alingment file selected.\n"  elif [ -a "$1" ]  then    echo Input file: $1    if [ -a $1.bai ]    then      if [ -z "$2" ]      then         echo -e "Organism : Human (hg19)"      else        if [ $2 == "mouse" ]        then          echo -e "Organism : Mouse (mm9)"          # This is Mouse GAPDH                   REGION="chr6:125111870-125115791"         elif [ $2 == "human" ]         then           echo -e "Organism : Human (hg19)"         else           echo -e "GTF file : $2"           if [ -a $2 ]           then            REGION="`cut -f1 $2 | uniq | head -n 3`"          else            echo -e "\nCouldn\'t find the GTF file.\n"             exit          fi        fi      fi      STRANDED="YES"      echo      for R in `echo $REGION`      do        SUB_TOTAL=`samtools view -f 0x40 $1 $R | wc -l`        if [ $SUB_TOTAL == "0" ]        then          echo -e "\nYour BAM file and the oranism are not matched.\n"          exit        fi        SUB_NEG=`samtools view -f 0x40 $1 $R | grep XS:A:- | wc -l`        SUB_POS=`samtools view -f 0x40 $1 $R | grep XS:A:+ | wc -l`        SUB_DOM=$SUB_NEG         MAJOR=neg-strand        if [ $SUB_POS -gt $SUB_DOM ]        then          SUB_DOM=$SUB_POS          MAJOR=pos-strand        fi        RATIO=`echo "scale = 2; $SUB_DOM * 100 / $SUB_TOTAL"| bc`        echo $RATIO% of reads \($SUB_DOM\/$SUB_TOTAL\) are $MAJOR in $R        if [ "$(echo $RATIO '<' 90.00 | bc -l)" -eq 1 ]        then          STRANDED="NO"        fi      done      if [ $STRANDED == "YES" ]      then        echo -e "\nThe data may be stranded."      else        echo -e "\nThe data looks like un-stranded."      fi    else      echo -e "\nNo index file found, quitting...\n"    fi  fi