Experiment / E5O2W2XZ6Episomal Plasmid MPRA

PYS-2 MPRA of model-guided and randomized ancestral CRE reconstitution trajectories

Retracing and rewriting the evolutionary trajectories of mammalian developmental enhancers

A separate episomal 5′ MPRA tested sequential intermediates generated by introducing mouse-derived substitutions and indels into the inferred common mammalian ancestor (Anc239) of the Gata4, Epas1, and Lama1 enhancer tiles. Each trajectory was either ChromBPNet-model ordered or one of ten random mutation orders, with six biological replicates represented by labels 1a, 1b, 2a, 2b, 3a, and 3b.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: CVCL:3776
Reference genome: GRCm39
Design focus: Variant-focused
Region of interest: chr14:63468603-63468903,chr17:67961106-67961406,chr17:87048616-87048916

Perturbation & assay details

Basal / Untreated

Bulk episomal 5′ reporter assay with each synthetic intermediate 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 converted to activity scores; the deposited data include model-guided and random-order trajectories plus controls (IGVFDS9730EWQE; files IGVFFI3560GEAZ, IGVFFI3464DNIG, and IGVFFI1341AKRF).

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 · 86 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: Focal CRE identifier parsed from the design name.
design_group: Parsed design class: model_guided_reconstitution, random_reconstitution, or control.
control_type: Active or inactive control label when applicable.
trajectory_objective: Model-guided or random-order trajectory objective.
trajectory_step: Mutation step/rank parsed from the design name.
trajectory_iteration: Random-trajectory iteration when encoded in the design name.
mutation_type: Mutation event type: substitution, insertion, or deletion when encoded.
ortholog_species_or_node: Ancestral background node encoded in the design name, typically fullTreeAnc239.
trajectory_recipient: Recipient species encoded in the design name, typically Mus_musculus.
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_rep1a: Raw DNA barcode-UMI count for replicate 1a.
rna_counts_rep1a: Raw RNA barcode-UMI count for replicate 1a.
dna_normalized_rep1a: Library-normalized DNA count for replicate 1a.
rna_normalized_rep1a: Library-normalized RNA count for replicate 1a.
activity_ratio_rep1a: RNA-normalized divided by DNA-normalized count for replicate 1a.
log2_activity_rep1a: Deposited log2 RNA/DNA activity score for replicate 1a.
n_bc_rep1a: Number of measured barcodes for replicate 1a.
dna_counts_rep1b: Raw DNA barcode-UMI count for replicate 1b.
rna_counts_rep1b: Raw RNA barcode-UMI count for replicate 1b.
dna_normalized_rep1b: Library-normalized DNA count for replicate 1b.
rna_normalized_rep1b: Library-normalized RNA count for replicate 1b.
activity_ratio_rep1b: RNA-normalized divided by DNA-normalized count for replicate 1b.
log2_activity_rep1b: Deposited log2 RNA/DNA activity score for replicate 1b.
n_bc_rep1b: Number of measured barcodes for replicate 1b.
dna_counts_rep2a: Raw DNA barcode-UMI count for replicate 2a.
rna_counts_rep2a: Raw RNA barcode-UMI count for replicate 2a.
dna_normalized_rep2a: Library-normalized DNA count for replicate 2a.
rna_normalized_rep2a: Library-normalized RNA count for replicate 2a.
activity_ratio_rep2a: RNA-normalized divided by DNA-normalized count for replicate 2a.
log2_activity_rep2a: Deposited log2 RNA/DNA activity score for replicate 2a.
n_bc_rep2a: Number of measured barcodes for replicate 2a.
dna_counts_rep2b: Raw DNA barcode-UMI count for replicate 2b.
rna_counts_rep2b: Raw RNA barcode-UMI count for replicate 2b.
dna_normalized_rep2b: Library-normalized DNA count for replicate 2b.
rna_normalized_rep2b: Library-normalized RNA count for replicate 2b.
activity_ratio_rep2b: RNA-normalized divided by DNA-normalized count for replicate 2b.
log2_activity_rep2b: Deposited log2 RNA/DNA activity score for replicate 2b.
n_bc_rep2b: Number of measured barcodes for replicate 2b.
dna_counts_rep3a: Raw DNA barcode-UMI count for replicate 3a.
rna_counts_rep3a: Raw RNA barcode-UMI count for replicate 3a.
dna_normalized_rep3a: Library-normalized DNA count for replicate 3a.
rna_normalized_rep3a: Library-normalized RNA count for replicate 3a.
activity_ratio_rep3a: RNA-normalized divided by DNA-normalized count for replicate 3a.
log2_activity_rep3a: Deposited log2 RNA/DNA activity score for replicate 3a.
n_bc_rep3a: Number of measured barcodes for replicate 3a.
dna_counts_rep3b: Raw DNA barcode-UMI count for replicate 3b.
rna_counts_rep3b: Raw RNA barcode-UMI count for replicate 3b.
dna_normalized_rep3b: Library-normalized DNA count for replicate 3b.
rna_normalized_rep3b: Library-normalized RNA count for replicate 3b.
activity_ratio_rep3b: RNA-normalized divided by DNA-normalized count for replicate 3b.
log2_activity_rep3b: Deposited log2 RNA/DNA activity score for replicate 3b.
n_bc_rep3b: Number of measured barcodes for replicate 3b.
replicate_count: Number of activity replicates included in the row.
mean_dna_counts: Arithmetic mean raw DNA count across six replicates.
mean_rna_counts: Arithmetic mean raw RNA count across six replicates.
mean_dna_normalized: Arithmetic mean normalized DNA count across six replicates.
mean_rna_normalized: Arithmetic mean normalized RNA count across six replicates.
mean_n_bc: Arithmetic mean measured-barcode count across six replicates.
min_n_bc: Minimum measured-barcode count across six 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, 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 six listed replicates; RNA counts were not thresholded so genuinely inactive intermediates were preserved. The processed table retains 2,633 of 2,956 scored count-table elements: 258 model-guided reconstitution elements, 2,364 random reconstitution elements, and 11 controls. One hundred sixty count-table identifiers lacked a corresponding public sequence design and 163 additional elements failed the all-replicate barcode-coverage filter. Pairwise Spearman correlations across retained non-control log2 activity scores ranged from 0.818 to 0.848.

Curation notes

This is the separate evolutionary reconstitution library (IGVFDS9730EWQE). The deposited count table has six labels per element (1a, 1b, 2a, 2b, 3a, 3b), consistent with the supplementary analysis describing six biological replicates. The public sequence-design file does not contain 160 identifiers that appear in the score/count table; those rows were excluded because their sequence could not be verified. Reconstitution constructs include naturally variable lengths because ancestral-to-mouse indels are part of the design.

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