SV40-promoter episomal MPRA of FAM13A COPD-locus variants in Beas-2B cells
Identification of Functional Variants in the FAM13A Chronic Obstructive Pulmonary Disease Genome-Wide Association Study Locus by Massively Parallel Reporter AssaysA library of 144-bp synthetic oligonucleotides carrying both alleles for common variants in and near FAM13A was cloned upstream of an SV40 promoter and assayed in Beas-2B human bronchial epithelial cells. Barcode sequencing of the input plasmid library and poly(A) RNA output was used to quantify allele- and context-specific regulatory activity.
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.
Perturbation & assay details
Basal / Untreated
The pGL4.10M2 episomal MPRA library contained 144-bp oligonucleotides representing both alleles for 606 SNPs selected from COPD GWAS and FAM13A eQTL evidence, with the variant placed at left, center, or right positions and in forward or reverse-complement orientation. The oligos were cloned with unique 11-nt barcodes upstream of the stronger SV40 promoter, transfected into Beas-2B cells using a Nucleofector II device, and harvested 24 hours later; the input library was PCR-amplified and the output poly(A) RNA was reverse-transcribed and barcode-amplified for 36-nt single-end Illumina HiSeq 2000 sequencing. GEO counts were generated by exact barcode matching.
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 · 25 definitions
- element_id
- Original GEO oligo identifier for one allele, SNP location, and oligonucleotide orientation.
- snp_id
- dbSNP rs identifier for the tested variant.
- allele
- Allele carried by the synthetic oligonucleotide.
- chromosome
- Chromosome reported by GEO.
- position_hg19
- 1-based SNP coordinate on the hg19 reference assembly.
- snp_location
- Position of the SNP within the 144-bp oligo: L (left), C (center), or R (right).
- orientation
- Oligo orientation relative to the reference genome: F (forward) or RC (reverse complement).
- promoter
- Reporter promoter used in this experiment (SV40).
- input_sample
- GEO sample accession for the input plasmid-library barcode counts.
- rna_sample
- GEO sample accession for the poly(A) RNA barcode counts.
- input_sra
- SRA experiment accession corresponding to the input library.
- rna_sra
- SRA experiment accession corresponding to the RNA output library.
- barcode_count_design
- Number of barcodes assigned to the oligo in the released design dictionary.
- barcode_count_observed
- Number of barcode rows observed for the oligo in the released count matrix.
- barcode_count_qc
- Number of observed barcodes retained after requiring input DNA count >=10.
- dna_count_sum
- Sum of input DNA reads over QC-passing barcodes.
- rna_count_sum
- Sum of RNA output reads over QC-passing barcodes.
- dna_count_mean
- Mean input DNA reads per QC-passing barcode.
- rna_count_mean
- Mean RNA output reads per QC-passing barcode.
- median_log2_normalized_rna_dna
- Median per-barcode log2 RNA/DNA activity after adding a 0.5 pseudocount and applying the library-size offset log2(total input DNA / total RNA).
- aggregate_log2_normalized_rna_dna
- Aggregate log2 RNA/DNA activity using QC-passing summed counts, a 0.5 pseudocount, and the same library-size offset.
- log2_activity_iqr
- Interquartile range of the per-barcode normalized log2 RNA/DNA activity values.
- allelic_effect_log2_hl
- Median of all pairwise differences between this allele's and the other allele's per-barcode normalized log2 RNA/DNA activities within the same SNP, oligo location, and orientation; positive values indicate higher activity for the row allele.
- allelic_effect_pvalue
- Two-sided normal-approximation Mann-Whitney p-value comparing the two allele barcode activity distributions within the same SNP, location, and orientation; blank when a paired contrast is unavailable.
- allelic_effect_fdr
- Benjamini-Hochberg adjustment of the barcode-level allele p-values across paired contexts in this experiment; the same value is repeated on both allele rows.
Quality control
The GEO record documents exact matching of barcode-dictionary entries to sequencing reads. Package QC verified that all 218,526 observed barcode rows mapped uniquely to the 7,270 design oligos with no barcode/design mismatches, removed barcode observations with input DNA count <10, and retained oligos only when at least 5 barcodes passed that threshold; zero RNA counts were retained and given a 0.5 pseudocount for activity ratios. This produced 7,138 of 7,270 oligos for the SV40 table. GEO exposes one input and one RNA sample for this promoter, so the allele p-values and Benjamini-Hochberg FDR values in the table are exploratory barcode-level Mann-Whitney estimates and were not used as a hard QC filter.
Curation notes
Beas-2B was resolved to Cellosaurus CVCL:0168 (the source Cellosaurus accession is written CVCL_0168). The paper reports 606 tested SNPs and 45 unique SNPs with significant allele-specific MPRA effects; this table retains all QC-passing tested oligos rather than only the published significant subset. The GEO release provides counts and barcode design but not the paper's derived significance tables, so the packaged activity and allele-effect columns are transparent calculations from the raw counts. Blank allele-effect fields indicate that the matched allele failed the package coverage filter. Raw sequencing reads were intentionally not included.