Experiment / E92DMK59CEpisomal Plasmid MPRA

Episomal MPRA of red-blood-cell trait variants in standard K562 cells

Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits

A 145-bp variant-focused oligonucleotide library representing major/reference and minor/alternate alleles across three variant-centered sliding windows was transfected into standard human K562 cells. Reporter RNA was measured 48 hours after transfection and normalized to plasmid DNA input across six RNA replicates.

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Organism: Human
Taxonomy ID: NCBITaxon:9606
Biosample: CVCL:0004
Reference genome: hg19
Design focus: Variant-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

The pMPRA1/minP-luc2 episomal reporter library contained approximately 145-bp genomic inserts centered on each tested variant, with reference and alternate alleles placed in left, middle, and right sliding windows. Each construct was assigned 14 designed 11-nt barcodes. The pooled plasmid library was introduced into K562 cells by nucleofection; plasmid DNA input was sequenced in two replicates and reporter RNA output was sequenced after 48 hours in six K562 control-condition replicates.

Processed data

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Column dictionary · 40 definitions

construct_id: Exact GEO construct name, including allele, sliding-window geometry, and source class.
variant_id: Stable package variant key in the form chr:position:reference>alternate; the GEO source did not provide dbSNP rs identifiers.
chromosome: hg19 chromosome without a chr prefix.
position_hg19: 1-based hg19 coordinate of the tested variant.
ref_allele: Reference allele reported in the GEO raw matrix.
alt_allele: Alternate allele reported in the GEO raw matrix.
allele_label: GEO construct allele label: Ref or Mut.
allele: Normalized allele label: reference for Ref, alternate for Mut.
clean_class: GEO source construct class: var or mod.
window: Variant-centered sliding-window label: left, middle, or right.
window_geometry: Fraction of the 145-bp insert on the 5-prime and 3-prime sides of the variant.
sequence_145bp: 145-bp genomic insert sequence for this allele and window.
n_barcodes_total: Number of designed barcodes for the construct; the source design assigns 14.
n_barcodes_qc: Number of barcodes with at least one DNA and one K562 RNA replicate meeting the raw-count >=8 eligibility threshold.
n_dna_observations_qc: Number of DNA barcode-replicate observations contributing to the normalized activity summary after the count threshold.
n_rna_observations_qc: Number of K562 RNA barcode-replicate observations contributing to the normalized activity summary after the count threshold.
dna_rep1_barcodes_count_ge_8: Number of the 14 barcode rows with K562_minP_DNA1 raw count >=8.
dna_rep2_barcodes_count_ge_8: Number of the 14 barcode rows with K562_minP_DNA2 raw count >=8.
dna_rep1_count_sum: Raw K562_minP_DNA1 counts summed over all 14 source barcode rows.
dna_rep2_count_sum: Raw K562_minP_DNA2 counts summed over all 14 source barcode rows.
rna_rep1_count_sum: Raw K562_CTRL_minP_RNA1 counts summed over all 14 source barcode rows.
rna_rep1_barcodes_count_ge_8: Number of the 14 barcode rows with K562_CTRL_minP_RNA1 raw count >=8.
rna_rep2_count_sum: Raw K562_CTRL_minP_RNA2 counts summed over all 14 source barcode rows.
rna_rep2_barcodes_count_ge_8: Number of the 14 barcode rows with K562_CTRL_minP_RNA2 raw count >=8.
rna_rep3_count_sum: Raw K562_CTRL_minP_RNA3 counts summed over all 14 source barcode rows.
rna_rep3_barcodes_count_ge_8: Number of the 14 barcode rows with K562_CTRL_minP_RNA3 raw count >=8.
rna_rep4_count_sum: Raw K562_CTRL_minP_RNA4 counts summed over all 14 source barcode rows.
rna_rep4_barcodes_count_ge_8: Number of the 14 barcode rows with K562_CTRL_minP_RNA4 raw count >=8.
rna_rep5_count_sum: Raw K562_CTRL_minP_RNA5 counts summed over all 14 source barcode rows.
rna_rep5_barcodes_count_ge_8: Number of the 14 barcode rows with K562_CTRL_minP_RNA5 raw count >=8.
rna_rep6_count_sum: Raw K562_CTRL_minP_RNA6 counts summed over all 14 source barcode rows.
rna_rep6_barcodes_count_ge_8: Number of the 14 barcode rows with K562_CTRL_minP_RNA6 raw count >=8.
dna_mean_log2_cpm: Mean pseudocount-adjusted log2 DNA CPM across eligible barcode-level DNA observations.
rna_mean_log2_cpm: Mean pseudocount-adjusted log2 K562 RNA CPM across eligible barcode-level RNA observations.
log2_rna_dna_activity: Construct activity, calculated as rna_mean_log2_cpm minus dna_mean_log2_cpm.
activity_sd_across_barcodes: Sample standard deviation of eligible barcode-level log2 RNA/DNA activities.
activity_se_across_barcodes: Standard error of the eligible barcode-level log2 RNA/DNA activity mean.
allelic_log2_fc_alt_minus_ref: Package-derived alternate-construct activity minus reference-construct activity for the same variant, window, and source class; blank if the paired construct did not pass QC.
allelic_pair_qc: TRUE when both reference and alternate constructs in the same variant/window/source-class pair passed QC.
qc_pass: TRUE for every construct retained in this processed table after the package QC filter.

Quality control

The authors added a pseudocount of 1, normalized DNA and RNA barcode counts to counts per million (CPM), log2-transformed the values, removed barcode observations below the reported count threshold of 8 per replicate, and assessed allelic effects with two-sided Mann-Whitney U tests using FDR <1%. For this package, a barcode was eligible for this condition when at least one of the two DNA replicates and at least one of the six K562 RNA replicates had a raw count >=8; a construct was retained only when at least 5 of its 14 barcodes were eligible. The table contains 15,015 retained constructs from the 16,534 GEO source constructs; all rows have qc_pass=TRUE. Construct activity is the mean barcode-level log2 RNA/DNA score across eligible barcodes, and the paired alternate-minus-reference effect is populated only when both allele constructs pass QC.

Curation notes

The GEO matrix is a processed barcode-count source rather than raw FASTQ. The design and barcode-mapping files are retained in raw_data and validate the 14-barcode construct layout. Source coordinates are hg19 and the source matrix does not include rs identifiers, so variant_id preserves the coordinate and allele identity. Source-marked mod constructs are retained because they are part of the published library. Shared plasmid-DNA replicates are used for both condition-specific experiments. The package-derived activity and paired allelic effect are intended as clean research summaries and are not claimed to reproduce the paper's fitted FDR statistics exactly.

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