Bioinformatics procedures
HT-5Pseq reads were trimmed with a 3’-sequencing adapter using cutadapt V1.16 (http://gensoft.pasteur.fr/docs/cutadapt/1.6/index.html). The 8-nt random barcodes on the 5 ̵́ ends of the reads were extracted and added to the header of the fastq file as the UMI employing UMI-tools. 5‘P reads were mapped to S. cerevisiae (SGD R64-1-1) using STAR 2.7.0 with parameter –alignEndsType Extend5pOfRead1 to exclude soft-clipped bases on the 5 ̵́ end. After removing PCR duplications using UMI-tools, analysis of the 5 ̵́ ends positions was performed using the Fivepseq package (Nersisyan et al., 2020, http://pelechanolab.com/software/fivepseq), including the relative distance to start and stop codons. In particular, the unique 5´ mRNA reads within biological samples were summed and subsequently normalized to reads per million (rpm). Standalone normalized average density plots around genomic features were calculated with R and Python software ngs.plot v2.61 (Schen et al., 2014) using indexed alignment files as inputs and the internal SacCer3 database annotation as reference. The statistical robustness parameter, which filters out 0.5% of the genes with the most extreme (high and low) count values, was applied to all calculations.
5’Cap reads were processed as described for HT-5Pseq reads. In general, 5’Cap reads were trimmed using a 3’-sequencing adapter and the extracted 8-nt random barcodes were used as UMI. The 5’Cap reads were mapped toS. cerevisiae (SGD R64-1-1) using STAR 2.7.0. PCR duplicates were removed using means of UMI-tools.
Heatmaps and the accompanying average metaplots displaying HT-5Pseq alongside the 5’Capseq datasets were generated using the bamCoverage, computeMatrix and plotHeatmap functions from the deepTools2 package (Ramírez et al., 2026). The transcription start site (TSS) and polyA site (pA) annotations were taken from (Xu et al., 2009). Spearman correlation values and statistical test result asterisks were inserted into ggplot2-generated plots using the ggpubr package (https://rpkgs.datanovia.com/ggpubr).