Experiment / E0SHYCWK65′ UTR / Translation Efficiency MPRA (MPTA)

HEK293 5′ UTR allele translation MPRA

Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

An episomal plasmid library containing 1,507 unique 5′ UTR sequence-context reporters representing 997 neurodevelopmental-disorder-family mutations was transiently transfected into human HEK293 cells. Each reference and alternate allele was represented by ten barcodes, and DNA, total RNA, 40S, 80S, polysome, TRAP, and Maxi-library counts were collected across six biological replicates.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0045
Reference genome: GRCh38
Design focus: Variant-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

Episomal CMV-driven tdTomato reporter with the tested 5′ UTR sequence between the promoter and reporter ORF and a barcode in the transcribed reporter region. Ribosome-density measurements used 40S, 80S/monosome, and polysome-associated RNA; total RNA/DNA measured reporter abundance, and TRAP and Maxi samples are retained as additional library readouts.

Processed data

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Column dictionary · 100 definitions

reporter_context_id: Normalized identifier for a unique variant/transcript sequence context in the MPRA library.
variant_id: Chromosome and coordinate portion of the tested variant identifier.
chromosome: Human chromosome for the tested variant.
start: Annotated 1-based variant start coordinate.
end: Annotated 1-based variant end coordinate.
strand: Annotated transcript strand.
reference_allele: Reference allele used in the reference reporter.
alternate_allele: Alternate allele used in the mutant reporter.
family_id: Neurodevelopmental-disorder family identifier from the source library.
sample_id: Source sample/context identifier.
gene_id: Ensembl gene identifier.
gene_symbol: Gene symbol.
phenotype: Source variant class, case or control.
transcript_accession: Transcript accession used for sequence annotation.
transcript_id: Ensembl transcript identifier represented by the reporter.
duplicate_ids: Source duplicate-context identifiers, when present.
reference_sequence: Reference 5′ UTR reporter sequence inserted into the construct.
alternate_sequence: Alternate 5′ UTR reporter sequence inserted into the construct.
reference_sequence_length: Length in nucleotides of the reference reporter sequence.
alternate_sequence_length: Length in nucleotides of the alternate reporter sequence.
relative_variant_start: Variant start relative to the sequence context.
relative_variant_end: Variant end relative to the sequence context.
relative_coding_start: Coding-start position relative to the sequence context.
final_atg_position: Position of the final canonical ATG in the designed reporter context.
final_rf_shift: Source reading-frame adjustment for the designed context.
reference_mfe: Predicted minimum free energy of the reference sequence.
alternate_mfe: Predicted minimum free energy of the alternate sequence.
delta_mfe: Source alternate-minus-reference MFE difference.
reference_uorf_count: Predicted/annotated upstream open-reading-frame count in the reference sequence.
alternate_uorf_count: Predicted/annotated upstream open-reading-frame count in the alternate sequence.
uorf_change: Source indicator of an upstream ORF gain/loss or change.
uaug_context_perturbed: Indicator that the variant perturbs an upstream AUG context.
any_gtex_brain_expressed: Indicator that any represented transcript is expressed in GTEx brain data.
pli: Probability of loss-of-function intolerance annotation.
sfari_gene_score: SFARI gene evidence score.
sfari_gene_syndromic: SFARI syndromic-gene indicator.
sfari_gene_denovo: SFARI de novo-gene indicator.
designed_barcodes_reference: Number of reference-allele barcodes present in the source count matrix/library design.
designed_barcodes_alternate: Number of alternate-allele barcodes present in the source count matrix/library design.
observed_barcodes_reference: Number of reference barcode rows retained in the GEO matrix for this context.
observed_barcodes_alternate: Number of alternate barcode rows retained in the GEO matrix for this context.
significant_metric_count_q05: Number of the six Table S2 comparisons with author q-value below 0.05.
qc_pass: Package QC flag; true for contexts retained after the paper’s barcode/replicate QC.
qc_umi_threshold: UMI threshold used in the barcode count audit, 20 counts per fraction.
qc_replicates_total_rna_dna: Number of biological replicates with at least three reference and three alternate barcodes passing the 20-UMI threshold for total RNA/DNA.
qc_ref_barcodes_min_total_rna_dna: Minimum passing reference barcode count across qualifying total RNA/DNA replicates.
qc_alt_barcodes_min_total_rna_dna: Minimum passing alternate barcode count across qualifying total RNA/DNA replicates.
qc_replicates_eighty_s_forty_s: Number of qualifying replicates for the 80S/40S count audit.
qc_ref_barcodes_min_eighty_s_forty_s: Minimum passing reference barcode count for the 80S/40S audit.
qc_alt_barcodes_min_eighty_s_forty_s: Minimum passing alternate barcode count for the 80S/40S audit.
qc_replicates_eighty_s_polysome: Number of qualifying replicates for the 80S/polysome count audit.
qc_ref_barcodes_min_eighty_s_polysome: Minimum passing reference barcode count for the 80S/polysome audit.
qc_alt_barcodes_min_eighty_s_polysome: Minimum passing alternate barcode count for the 80S/polysome audit.
qc_replicates_polysome_total_rna: Number of qualifying replicates for the polysome/total-RNA count audit.
qc_ref_barcodes_min_polysome_total_rna: Minimum passing reference barcode count for the polysome/total-RNA audit.
qc_alt_barcodes_min_polysome_total_rna: Minimum passing alternate barcode count for the polysome/total-RNA audit.
qc_replicates_forty_s_total_rna: Number of qualifying replicates for the 40S/total-RNA count audit.
qc_ref_barcodes_min_forty_s_total_rna: Minimum passing reference barcode count for the 40S/total-RNA audit.
qc_alt_barcodes_min_forty_s_total_rna: Minimum passing alternate barcode count for the 40S/total-RNA audit.
qc_replicates_eighty_s_total_rna: Number of qualifying replicates for the 80S/total-RNA count audit.
qc_ref_barcodes_min_eighty_s_total_rna: Minimum passing reference barcode count for the 80S/total-RNA audit.
qc_alt_barcodes_min_eighty_s_total_rna: Minimum passing alternate barcode count for the 80S/total-RNA audit.
mean_umi_ref_dna: Mean unnormalized DNA UMI count across reference barcode rows and six replicates.
mean_umi_alt_dna: Mean unnormalized DNA UMI count across alternate barcode rows and six replicates.
mean_umi_ref_total_rna: Mean unnormalized total-RNA UMI count for reference barcode rows.
mean_umi_alt_total_rna: Mean unnormalized total-RNA UMI count for alternate barcode rows.
mean_umi_ref_40s: Mean unnormalized 40S UMI count for reference barcode rows.
mean_umi_alt_40s: Mean unnormalized 40S UMI count for alternate barcode rows.
mean_umi_ref_80s: Mean unnormalized 80S UMI count for reference barcode rows.
mean_umi_alt_80s: Mean unnormalized 80S UMI count for alternate barcode rows.
mean_umi_ref_polysome: Mean unnormalized polysome UMI count for reference barcode rows.
mean_umi_alt_polysome: Mean unnormalized polysome UMI count for alternate barcode rows.
mean_umi_ref_trap: Mean unnormalized TRAP-associated UMI count for reference barcode rows.
mean_umi_alt_trap: Mean unnormalized TRAP-associated UMI count for alternate barcode rows.
mean_umi_ref_maxi: Mean unnormalized Maxi-prepped plasmid-library UMI count for reference barcode rows.
mean_umi_alt_maxi: Mean unnormalized Maxi-prepped plasmid-library UMI count for alternate barcode rows.
total_rna_dna_logfc: Author-reported allelic log2 fold change for Table S2.1 Total RNA-DNA Enrichment.
total_rna_dna_empirical_pvalue: Author empirical p-value for the Table S2.1 total RNA-DNA allelic effect.
total_rna_dna_qvalue: Author multiple-testing-corrected q-value for Table S2.1.
total_rna_dna_significant_q05: Derived true when total_rna_dna_qvalue is below 0.05.
eighty_s_forty_s_logfc: Author-reported allelic log2 fold change for Table S2.2 80S-40S Enrichment.
eighty_s_forty_s_empirical_pvalue: Author empirical p-value for Table S2.2.
eighty_s_forty_s_qvalue: Author multiple-testing-corrected q-value for Table S2.2.
eighty_s_forty_s_significant_q05: Derived true when eighty_s_forty_s_qvalue is below 0.05.
eighty_s_polysome_logfc: Author-reported allelic log2 fold change for Table S2.3 80S-Polysome Enrichment; source orientation is preserved.
eighty_s_polysome_empirical_pvalue: Author empirical p-value for Table S2.3.
eighty_s_polysome_qvalue: Author multiple-testing-corrected q-value for Table S2.3.
eighty_s_polysome_significant_q05: Derived true when eighty_s_polysome_qvalue is below 0.05.
polysome_total_rna_logfc: Author-reported allelic log2 fold change for Table S2.4 Polysome-Total RNA Enrichment.
polysome_total_rna_empirical_pvalue: Author empirical p-value for Table S2.4.
polysome_total_rna_qvalue: Author multiple-testing-corrected q-value for Table S2.4.
polysome_total_rna_significant_q05: Derived true when polysome_total_rna_qvalue is below 0.05.
forty_s_total_rna_logfc: Author-reported allelic log2 fold change for Table S2.5 40S-Total RNA Enrichment.
forty_s_total_rna_empirical_pvalue: Author empirical p-value for Table S2.5.
forty_s_total_rna_qvalue: Author multiple-testing-corrected q-value for Table S2.5.
forty_s_total_rna_significant_q05: Derived true when forty_s_total_rna_qvalue is below 0.05.
eighty_s_total_rna_logfc: Author-reported allelic log2 fold change for Table S2.6 80S-Total RNA Enrichment.
eighty_s_total_rna_empirical_pvalue: Author empirical p-value for Table S2.6.
eighty_s_total_rna_qvalue: Author multiple-testing-corrected q-value for Table S2.6.
eighty_s_total_rna_significant_q05: Derived true when eighty_s_total_rna_qvalue is below 0.05.

Quality control

The authors removed barcode observations with fewer than 20 UMI counts in either fraction of a ratiometric measurement, required at least three biological replicates with at least three reference and three alternate barcodes after filtering, fit barcode-level log-ratio mixed models, and used blank-control empirical p-values with q-value correction. The package retains all 1,507 contexts because all six reported HEK comparisons pass the count-based replicate/barcode audit; q<0.05 flags are derived from the author’s Table S2 q-values.

Curation notes

The table is one row per tested variant/transcript sequence context, not one row per individual barcode. Source Table S2 logFC values are preserved in the orientation named by each supplemental sheet; no sign reorientation was applied. The GRCh38 reference assembly is supported by the authors’ public VEP analysis files.

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