PeakPrime: a peak-guided primer design pipeline for target enrichment in 3'-end RNA-seq

Targeted enrichment can offset the bias and depth requirements of random-primed second-strand synthesis in 3'-end RNA-seq by reallocating reads to transcripts of interest. We present PeakPrime, a reproducible Nextflow pipeline that (i) identifies high-coverage 3 ${ }^{\text{'}}$ RNA-seq regions via MACS2, (ii) selects exonic windows around significant peaks, (iii) designs strand-appropriate cDNA primers via Primer3, and (iv) screens specificity with Bowtie2, producing publication-ready plots and quality control (QC) summaries.

We demonstrate coverage-informed primer design using a custom 3 ${ }^{\text{'}}$ protocol that employs a barcoded oligo(dT)-VN primer, optimize parameters on IMR-32 neuroblastoma and Universal Human Reference RNA data, and validate primers by quantitative PCR (qPCR) and targeted 3 ${ }^{\text{'}}$ RNA-seq. Targeted libraries preserve gene-level expression correlations with random-primed 3 ${ }^{\text{'}}$ RNA-seq (Pearson $r\approx 0.88$ across conditions), while boosting the fraction of reads assigned to target genes from <1% to $\sim$ 25%-42% and increasing detection of the lowest-abundance transcripts. While empirically tested in bulk RNA-seq, PeakPrime enables extensions to single-cell and spatial workflows where oligo(dT) capture is followed by in situ reverse transcription and second-strand synthesis.

PeakPrime source code, example configs, and documentation are available at https://github.com/OncoRNALab/PeakPrime.