biostudies-literatureGuzman CNIDDK NIH HHSNIDA NIH HHSNIAID NIH HHSUC San DiegoNHGRI NIH HHSNIHNIH HHSNIGMS NIH HHSe80https://www.ebi.ac.uk/biostudies/studies/S-EPMC10450201biostudies-literatureUnknown51(15)Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique repoNucleic acids researchCombining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.PMC10450201R01 GM129523F31 HG011823U01DA051972U01 DA051972R01 GM134366R00 GM135515R21DA056177R21 DA056177R00GM135515R01GM134366U01 AI150748U01AI150748P30DK120515P30DK063491P30 DK063491P30 DK120515R01GM129523De Arruda Saldanha CDownes NLDuttke SZhu YBenner CGuzman CHeinz SfalseCombining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique repo2023-01-01T00:00:00Z2023 Aug2026-04-08T12:31:40.582Z2025-04-07T10:22:11.373ZS-EPMC104502013740379610.1093/nar/gkad562