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In the options, forward means the strand of the read must be the same as the strand of the transcript while reverse means the read must be the complementary strand to the transcript (Figure 3). The dash separates first- and second-in-pair. For paired end reads, we determine these by the flag information of the read in the BAM file. For single end reads, they are treated as the first read of paired end read. Briefly, the Strand specificity options are:

• No: Reads will be included in the calculation as long as they map to exonic regions, regardless of the direction.

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If the Report unexplained regions check button is selected, an additional report will be generated on the reads that are considered not compatible with any transcripts in the annotation provided. Based on the Min reads for unexplained region cutoff, the adjacent regions that meet the criteria are combined and region start and stop information will be reported.

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The bar chart displaying the distribution of raw read counts is helpful in assessing the expression level distribution within each sample. The X-axis is the read count range, Y axis is the number of features within the range, each bar is a sample. Hovering your mouse over the bar displays the following information (Figure 6):

• Sample name

• Range of read counts, “[ “represent inclusive, “)” represent exclusive, e.g. [0,0] means 0 read counts; (0,10] means the range is greater than 0 count but less than and equal to 10 counts.

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