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).