TAPAS analysis

TAPAS, a recently devised software, enabled us to detect which is the tool for detecting such alternative (or all) polyadenylation sites within a gene from RNAseq data³⁴. To run TAPAS, two types of file are required; one reference annotation file (ftp://hgdownload.soe.ucsc.edu/goldenPath/macFas5/database/refFlat.txt), which was downloaded from the UCSC database, and another one called the read coverage file that was generated from the BAM file using samtools (the program that utilizes SAM file) command upon the Linux operating system. The BAM file (.bam), which contained information about the read sequence, was generated from the RNA sequencing data (unpublished) of 30 cynomolgus macaques’ blood samples (Supplementary Fig. ^S5)⁴². These healthy macaques were from the NPRC of the KRIBB.

The reference annotation file and read coverage file were used as input files for TAPAS analyses. The output file consisted of six columns: gene name, chromosome name, the strand of the gene, detected APA sites, the abundance of those APA sites, and read count³⁴. We sorted and tried to extract the information from the output file for the 10 genes that had 3′UTR-end AluYRa1s, but we could extract information on only seven genes because of the absence of information in the reference gene file for the remaining 3 genes (CMBL, SLC16A14, PDK4). Another group of three genes (BLOC1S6, UBE2B, PAICS) was also excluded from the TAPAS analysis because of their different direction or inappropriate gene structure. Therefore, we extracted APA information for four genes (TK2, GTPBP4, PEX26, IRF9) from the TAPAS output file and conducted the statistical analysis.