Experiment / E6VXMPOS1Episomal Plasmid MPRA

PYS-2 MPRA of mammalian CRE orthologs and endogenous CRE tuning trajectories

Retracing and rewriting the evolutionary trajectories of mammalian developmental enhancers

A pooled episomal 5′ MPRA in mouse PYS-2 cells tested 1,871 extant or reconstructed mammalian orthologs of five 300-bp mouse developmental enhancer tiles together with model-nominated enhancement and ablation trajectories for those enhancers. The deposited library also contained promoter standards and other controls, and was measured in three biological replicates.

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Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.

Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CVCL:3776
Reference genome: GRCm39
Design focus: Other
Region of interest: chr4:111340574-111340874,chr11:55319627-55319927,chr14:63468603-63468903,chr17:67961106-67961406,chr17:87048616-87048916

Perturbation & assay details

Basal / Untreated

Bulk episomal 5′ reporter assay with CRE inserts upstream of a minimal promoter driving GFP and a 15-bp random molecular barcode in the reporter 5′ UTR. PYS-2 cells were transfected with Lipofectamine 2000, harvested 48 hours later, and DNA/RNA barcode-UMI counts were used to estimate RNA/DNA activity; three biological replicates were sequenced on NextSeq 2000. This IGVF data set combines the five-CRE ortholog library and the 50-step model-nominated enhancement/ablation libraries in one experiment (IGVFDS1754MDNZ; files IGVFFI3295ZQBJ, IGVFFI7026BKIY, and IGVFFI8148RLWG).

Processed data

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Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.

Column dictionary · 65 definitions

element_id: Element identifier used by the IGVF count and score tables.
sequence_design_name: Original sequence-design name.
sequence_design_quantification_id: Sequence-design quantification identifier; blank when not supplied.
sequence: Tested reporter insert sequence.
sequence_length: Length of the tested insert in nucleotides.
target_cre: One of the five focal CRE identifiers parsed from the design name; blank for controls without a focal CRE.
design_group: Parsed design class: ortholog, endogenous_tuning, or control.
control_type: Active or inactive control label when applicable.
trajectory_objective: For tuning constructs, enhancement or ablation objective.
trajectory_step: Mutation step parsed from the design name; blank for non-trajectory constructs.
trajectory_iteration: Trajectory iteration when encoded in the design name.
mutation_type: Mutation event type when encoded; blank for ortholog/tuning names without an event token.
ortholog_species_or_node: Species or reconstructed ancestral node encoded in the design name.
trajectory_recipient: Recipient species encoded for reconstitution designs; blank here except where present in source annotations.
sequence_design_category: Original IGVF sequence-design category.
sequence_design_class: Original IGVF sequence-design class, including test and control labels.
sequence_source: Source/provenance field from the sequence-design table.
sequence_info: Free-text design annotation from the sequence-design table.
design_reference: Reference assembly reported for the sequence design.
design_chromosome: Chromosome for designs with genomic coordinates.
design_start: Start coordinate for designs with genomic coordinates.
design_end: End coordinate for designs with genomic coordinates.
design_strand: Strand for designs with genomic coordinates.
variant_class: Original variant-class field; blank when not applicable.
variant_pos: Original variant-position field; blank when not applicable.
SPDI: Original SPDI variant representation; blank when not applicable.
allele: Original allele field; blank when not applicable.
dna_counts_rep1: Raw DNA barcode-UMI count for replicate 1.
rna_counts_rep1: Raw RNA barcode-UMI count for replicate 1.
dna_normalized_rep1: Library-normalized DNA count for replicate 1.
rna_normalized_rep1: Library-normalized RNA count for replicate 1.
activity_ratio_rep1: RNA-normalized divided by DNA-normalized count for replicate 1.
log2_activity_rep1: Deposited log2 RNA/DNA activity score for replicate 1.
n_bc_rep1: Number of measured barcodes for replicate 1.
dna_counts_rep2: Raw DNA barcode-UMI count for replicate 2.
rna_counts_rep2: Raw RNA barcode-UMI count for replicate 2.
dna_normalized_rep2: Library-normalized DNA count for replicate 2.
rna_normalized_rep2: Library-normalized RNA count for replicate 2.
activity_ratio_rep2: RNA-normalized divided by DNA-normalized count for replicate 2.
log2_activity_rep2: Deposited log2 RNA/DNA activity score for replicate 2.
n_bc_rep2: Number of measured barcodes for replicate 2.
dna_counts_rep3: Raw DNA barcode-UMI count for replicate 3.
rna_counts_rep3: Raw RNA barcode-UMI count for replicate 3.
dna_normalized_rep3: Library-normalized DNA count for replicate 3.
rna_normalized_rep3: Library-normalized RNA count for replicate 3.
activity_ratio_rep3: RNA-normalized divided by DNA-normalized count for replicate 3.
log2_activity_rep3: Deposited log2 RNA/DNA activity score for replicate 3.
n_bc_rep3: Number of measured barcodes for replicate 3.
replicate_count: Number of activity replicates included in the row.
mean_dna_counts: Arithmetic mean raw DNA count across replicates.
mean_rna_counts: Arithmetic mean raw RNA count across replicates.
mean_dna_normalized: Arithmetic mean normalized DNA count across replicates.
mean_rna_normalized: Arithmetic mean normalized RNA count across replicates.
mean_n_bc: Arithmetic mean measured-barcode count across replicates.
min_n_bc: Minimum measured-barcode count across replicates; all retained rows are at least 10.
mean_activity_ratio: Arithmetic mean of per-replicate normalized RNA/DNA activity ratios.
sd_activity_ratio: Sample standard deviation of per-replicate activity ratios.
mean_log2_activity: Arithmetic mean of deposited per-replicate log2 activity scores.
sd_log2_activity: Sample standard deviation of deposited per-replicate log2 activity scores.
score_log2_fold_change: IGVF element-level BCalm log2 fold-change score.
score_activity_ratio: Element-level activity ratio obtained as 2 raised to score_log2_fold_change.
score_input_count: IGVF element-level normalized input count used by the score calculation.
score_output_count: IGVF element-level normalized output count used by the score calculation.
score_minus_log10_pvalue: Negative log10 element-level p-value from the IGVF score table.
score_minus_log10_qvalue: Negative log10 element-level multiple-testing-adjusted q-value from the IGVF score table.

Quality control

The upstream MPRA pipeline filtered CRE–barcode associations at a minimum of 10 association reads, removed duplicate/low-quality barcode mappings, quantified UMI-normalized RNA/DNA activity with a 1% upper-tail winsorization, and used BCalm comparisons to negative controls. For this package, a row was retained only when its sequence design and element score were present, the sequence contained only A/C/G/T/N characters, and n_bc was at least 10 in each of the three replicates; RNA counts were not thresholded so true low-activity/ablated constructs were preserved. The processed table retains 2,380 of 2,404 scored count-table elements: 1,845 ortholog test elements, 504 endogenous-tuning test elements, and 31 controls. Replicate Spearman correlations for non-control log2 activity were 0.967 (1–2), 0.967 (1–3), and 0.962 (2–3).

Curation notes

The physical library and three-replicate data submission combines ortholog sequences and endogenous CRE-tuning trajectories, so they remain one experiment/table with design_group and trajectory fields separating them. Ortholog sequences are 300-bp maximum-activity tiles with naturally variable lengths in some recovered designs; full-length tested mouse/control constructs are retained where present. The IGVF portal labels the submitted file assembly GRCm39, while the paper's methods describe initial mm10 mapping. The related saturation-mutagenesis/sub-tiling MPRA is a separate companion study and is not represented as a child experiment here.

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