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If the Require junction reads to match introns check button is selected, only junction reads that overlap with exonic regions and match the skipped bases of an intron in the transcript will be included in the calculation. Otherwise, as long as the reads overlap within the exonic region, they will be counted. Detailed information about read compatibility can be found in the Understanding Reads tutorial white paper.

Some library preparations reverse transcribe the mRNA into double stranded cDNA, thus losing strand information. In this case, the total transcript count will include all the reads that map to a transcript location. Others will preserve the strand information of the original transcript by only synthesizing the first strand cDNA.  Thus, only the reads that have sense compatibility with the transcripts will be included in the calculation. We recommend verifying with the data source how the NGS library was prepared to ensure correct option selection.

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References

1. Xing Y, Yu T, Wu YN, Roy M, Kim J, Lee C. An expectation-maximization algorithm for probabilistic reconstructions of full-length isoforms from splice graphs. Nucleic Acids Res. 2006; 34(10):3150-60.
   
   
   

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