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We developed a pipeline for TMB estimation by coupling the information from RNA-seq reads with the gene structure from the genome. The exon number calculation based on RNA-seq data was implemented by calculating the total number of reads mapped to a gene based on the percentage of exonic base coverage. For each gene, we calculated the gene size by applying the formula gene size = 100\*(the sum of all exonic base covered by the reads mapped to the gene)/the number of mapped reads. To test the accuracy of this method, we used three independent RNA-seq datasets from different tissues and calculated the correlation between gene size and read count.

Gene expression estimation {#Sec14}
————————–

We obtained the FPKM value of protein coding genes in each dataset by the Cufflinks software (v2.2.0).

Identification of alternatively spliced genes {#
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