Experiment / E5LBEV8XFEpisomal Plasmid MPRA

Comparative episomal MPRA of human and mouse orthologous regulatory elements in embryonic stem cells

Cis and trans effects differentially contribute to the evolution of promoters and enhancers

A paired library of 144-bp human and mouse orthologous TSS/enhancer tiles was tested in HUES64 human embryonic stem cells and mouse embryonic stem cells. Three biological replicates per cellular environment measured reporter RNA relative to plasmid-DNA barcode representation, enabling native, cis, trans, and cis-trans interaction effects.

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Organism: NCBITaxon:9606;NCBITaxon:10090
Taxonomy ID: NCBITaxon:9606;NCBITaxon:10090
Biosample: CVCL:B199;CL:0002322
Reference genome: hg19;mm9
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

Twist-synthesized 200-bp oligos placed a 144-bp regulatory sequence, an 11-bp barcode, and cloning sequence upstream of GFP. Each selected TSS was represented by a TSS-overlapping tile and an upstream tile; each tile had 13 barcodes, while random negative controls had 3 and CMV positive controls had 60. The same plasmid library was transfected into HUES64 hESCs and mESCs, with three biological replicates in each environment. HUES64 biological replicates used three independent transfections per passage that were summed for analysis, whereas each mESC passage used one transfection. MPRAnalyze estimated activity from barcode RNA versus input-DNA counts and modeled native, cis, trans, and cis-trans interaction effects.

Processed data

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Column dictionary · 58 definitions

element_pair_id: Unique source-record identifier formed from the human and mouse element IDs, representative tile, and original selection species; the same underlying orthologous pair can have two records when selected from both species' TSS lists.
human_element_id: Author design identifier for the human member of the orthologous pair.
mouse_element_id: Author design identifier for the mouse member of the orthologous pair.
representative_tile: Representative 144-bp tile selected by the authors for both species: tile1 overlaps the annotated TSS and tile2 is the upstream tile selected when it was more active in both species.
human_sequence_144bp: 144-bp human regulatory sequence used in the reporter construct for the representative tile.
mouse_sequence_144bp: 144-bp mouse regulatory sequence used in the reporter construct for the representative tile.
human_chrom: Human chromosome reported for the TSS in hg19.
human_tss_start_hg19: Human TSS coordinate reported by the authors on hg19.
human_cage_id: FANTOM5 CAGE peak identifier/interval and strand for the human element.
human_biotype: Detailed human regulatory-element biotype assigned by the authors.
human_minimal_biotype: Collapsed human biotype used in comparative analyses: mRNA, lncRNA, eRNA, other, or no CAGE activity.
human_annotation: Human GENCODE gene or FANTOM5 peak annotation associated with the element.
human_stem_expression: Author-provided stem-cell expression measure for the human element.
mouse_chrom: Mouse chromosome reported for the TSS in mm9.
mouse_tss_start_mm9: Mouse TSS coordinate reported by the authors on mm9.
mouse_cage_id: FANTOM5 CAGE peak identifier/interval and strand for the mouse element.
mouse_biotype: Detailed mouse regulatory-element biotype assigned by the authors.
mouse_minimal_biotype: Collapsed mouse biotype used in comparative analyses: mRNA, lncRNA, eRNA, other, or no CAGE activity.
mouse_annotation: Mouse GENCODE gene or FANTOM5 peak annotation associated with the element.
mouse_stem_expression: Author-provided stem-cell expression measure for the mouse element.
original_species: Species from which the original TSS was selected in the authors' design.
pair_biotype: Author's paired minimal-biotype classification used for downstream analyses.
human_dna_barcode_count: Number of designed input-DNA barcodes for the human representative tile; expected to be 13.
human_dna_barcodes_ge10: Number of human representative-tile barcodes with input-DNA count >=10 in the plasmid-DNA library.
human_dna_barcode_coverage: Fraction of designed human representative-tile barcodes with input-DNA count >=10.
mouse_dna_barcode_count: Number of designed input-DNA barcodes for the mouse representative tile; expected to be 13.
mouse_dna_barcodes_ge10: Number of mouse representative-tile barcodes with input-DNA count >=10 in the plasmid-DNA library.
mouse_dna_barcode_coverage: Fraction of designed mouse representative-tile barcodes with input-DNA count >=10.
human_activity_hESC: MPRAnalyze MPRA activity estimate for the human sequence in HUES64 hESCs.
mESC_activity_human_sequence: MPRAnalyze MPRA activity estimate for the human sequence in mESCs.
hESC_activity_mouse_sequence: MPRAnalyze MPRA activity estimate for the mouse sequence in HUES64 hESCs.
mouse_activity_mESC: MPRAnalyze MPRA activity estimate for the mouse sequence in mESCs.
human_activity_hESC_q: MPRAnalyze q value for human-sequence activity in HUES64 hESCs versus the random-sequence activity null.
mESC_activity_human_sequence_q: MPRAnalyze q value for human-sequence activity in mESCs versus the random-sequence activity null.
hESC_activity_mouse_sequence_q: MPRAnalyze q value for mouse-sequence activity in HUES64 hESCs versus the random-sequence activity null.
mouse_activity_mESC_q: MPRAnalyze q value for mouse-sequence activity in mESCs versus the random-sequence activity null.
active_in_either_native_context: TRUE when the human sequence has q<0.05 in hESCs or the mouse sequence has q<0.05 in mESCs; recomputed from the supplied author activity q values.
native_log2fc_mouse_vs_human: Author native-effect log2 fold change comparing mouse sequence in mESCs with human sequence in hESCs; positive values indicate higher native activity for mouse.
native_fdr: MPRAnalyze FDR/q value for the native human-versus-mouse activity comparison.
native_call: Author's significant/no-significant native-effect call after empirical negative-control and effect-size thresholds.
native_direction: Author's direction-resolved native-effect call, including the higher-activity species when significant.
cis_log2fc_mouse_vs_human_hESC: Cis-effect log2 fold change comparing mouse and human sequences in HUES64 hESCs; positive values indicate higher mouse-sequence activity.
cis_fdr_hESC: MPRAnalyze FDR/q value for the HUES64 hESC cis comparison.
cis_log2fc_mouse_vs_human_mESC: Cis-effect log2 fold change comparing mouse and human sequences in mESCs; positive values indicate higher mouse-sequence activity.
cis_fdr_mESC: MPRAnalyze FDR/q value for the mESC cis comparison.
cis_call: Combined author cis-effect call across the HUES64 and mESC models.
cis_direction: Direction-resolved combined cis-effect call selected by the authors.
trans_log2fc_mESC_vs_hESC_human_sequence: Trans-effect log2 fold change for the human sequence between mESC and HUES64 environments; positive values indicate higher activity in mESCs.
trans_fdr_human_sequence: MPRAnalyze FDR/q value for the trans comparison of the human sequence.
trans_log2fc_mESC_vs_hESC_mouse_sequence: Trans-effect log2 fold change for the mouse sequence between mESC and HUES64 environments; positive values indicate higher activity in mESCs.
trans_fdr_mouse_sequence: MPRAnalyze FDR/q value for the trans comparison of the mouse sequence.
trans_call: Combined author trans-effect call across human and mouse sequences.
trans_direction: Direction-resolved combined trans-effect call selected by the authors.
cis_trans_interaction_log2fc: Author's cis-by-trans interaction log2 effect estimate for the paired sequences and cellular environments.
cis_trans_interaction_fdr: MPRAnalyze FDR/q value for the cis-by-trans interaction test.
cis_trans_interaction_call: Author's significant/no-significant cis-trans interaction call.
cis_trans_interaction_direction: Direction-resolved cis-trans interaction call when available.
qc_pass: TRUE for every row retained after the author-reported and independently rechecked barcode-coverage QC filters.

Quality control

Applied the authors' QC: adapter and Phred-quality trimming; exact barcode matching with the upstream constant sequence; removal of low-count barcodes; and retention of sequence pairs with at least 50% of designed barcodes represented at >=10 counts in the input DNA library. Differential calls use the authors' MPRAnalyze empirical control calibration (less than 10% of negative controls called significant) and minimum significant null-control effect-size thresholds. An independent recheck of the representative human and mouse tiles in the downloaded GEO index/count files found 13 designed barcodes per sequence and retained all 2,952 source rows because both members of every pair had at least 50% of their input-DNA barcodes at >=10 counts. No rows with failed QC are present in table.csv.

Curation notes

This is one comparative MPRA experiment with two cellular environments rather than two independent libraries: HUES64 human embryonic stem cells (Cellosaurus CVCL:B199) and unspecified mouse embryonic stem cells (Cell Ontology CL:0002322). The target_organism and biosample_id fields therefore contain two semicolon-separated standardized CURIEs, in human-then-mouse order. The assay tests orthologous regulatory regions, not allelic SNPs, so it is classified as Region-focused. The table retains all 2,952 pairs surviving the paper's input-DNA barcode representation filter, including pairs without significant activity; the paper subsequently restricted some analyses to active pairs. active_in_either_native_context is a convenience flag recomputed from the supplied q values and yields 1,655 rows in this archive output, while the publication narrative reports 1,644 active pairs. Coordinates are in the authors' legacy hg19/mm9 assemblies. Activity columns are MPRAnalyze alpha estimates; raw barcode counts and the authors' full effect tables are retained under raw_data for provenance. Positive cis effects indicate higher mouse-sequence activity; positive trans effects indicate higher activity in the mESC environment. The downloaded GEO count files contain the full barcode count export, including constructs outside the analyzed TSS subset; the GEO index and author result tables define the analyzed subset used here.

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