Bovine Vex-seq splicing variant validation in MAC-T cells
Functional impact of splicing variants in the elaboration of complex traits in cattleThe authors transfected the allele-resolved Vex-seq plasmid library into immortalized bovine mammary epithelial MAC-T cells in three biological replicates. The library contained positive controls, bovine GWAS candidates (Var.GWAS), and prediction-enriched bovine variants (Var.P); exon inclusion was quantified as PSI and the alternate-minus-reference change as ΔPSI.
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
Vex-seq used an episomal modified pcAT7-Glo1 reporter. Each synthesized construct contained a test exon of 13–98 nt carrying the REF or ALT allele, 50 nt of upstream intron and 20 nt of downstream intron, four sequence barcodes per test sequence, and invariant vector sequences containing MfeI/SpeI sites. The study subset comprised the positive-control, Var.GWAS, and Var.P portions of the pooled library. MAC-T cells were transfected in triplicate with 2 µg PL2 and 6 µL Lipofectamine 2000 per well and harvested 48 h later. UMI-tagged cDNA was sequenced; reads aligning to transcripts with or without the test exon were counted to calculate PSI (%), and ΔPSI is the ALT PSI minus REF PSI.
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 · 39 definitions
- dataset
- Source library stratum: Positive control, Var.GWAS, or Var.P.
- variant_id
- rsID for GWAS/prediction variants; deterministic control key for positive controls without an rsID.
- rsid
- dbSNP rs identifier; blank for positive controls without one.
- species
- Species represented by the tested construct.
- chromosome
- Chromosome reported in the source spreadsheet.
- position
- 1-based variant position reported in the source spreadsheet.
- ref_allele
- Reference allele in the Vex-seq construct.
- alt_allele
- Alternative allele in the Vex-seq construct.
- gene_symbol
- Gene symbol associated with the tested variant.
- ensembl_gene_id
- Ensembl gene identifier.
- variant_consequence
- Source/Ensembl variant consequence; positive-control annotations are joined from the bona fide SDV sheet when coordinates match.
- vex_seq_region
- Test-region genomic interval and orientation used for the Vex-seq construct.
- variant_strand
- Test-region strand parsed from the Vex-seq region field.
- spliceai_consequence
- SpliceAI consequence label, when available.
- spliceai_score
- SpliceAI score for the variant; positive-control values are taken from the corresponding control annotation when available.
- spliceai_position
- Relative position associated with the reported SpliceAI consequence.
- pangolin_score
- Signed Pangolin splice-strength score.
- pangolin_position
- Relative position associated with the Pangolin score.
- gerp_score
- GERP conservation score from the Var.GWAS or Var.P source sheet.
- positive_control_validation_method
- Previously reported validation method for a positive-control splice-disrupting variant; blank for study candidate variants.
- associated_breeds
- Semicolon-separated breeds associated with this rsID in Supplementary Data 5.
- associated_phenotypes
- Semicolon-separated phenotypes associated with this rsID in Supplementary Data 5.
- gwas_imputation_r2
- Semicolon-separated imputation r² values from Supplementary Data 5; entries follow the associated breed/phenotype records.
- gwas_alt_frequency
- Semicolon-separated ALT allele frequencies from Supplementary Data 5; entries follow the associated breed/phenotype records.
- gwas_effects
- Semicolon-separated breed|phenotype|effect records from Supplementary Data 5.
- gwas_effect_sem
- Semicolon-separated breed|phenotype|effect-SEM records from Supplementary Data 5.
- gwas_p_values
- Semicolon-separated breed|phenotype|p-value records from Supplementary Data 5.
- gwas_logp
- Semicolon-separated breed|phenotype|−log10(p) records from Supplementary Data 5.
- cell_line
- MAC-T immortalized bovine mammary epithelial cell line used for this child experiment (Cellosaurus CVCL:U226).
- ref_psi_mean
- Mean percent-spliced-in (PSI) for the REF allele across the three MAC-T transfection replicates.
- alt_psi_mean
- Mean percent-spliced-in (PSI) for the ALT allele across the three MAC-T transfection replicates.
- delta_psi
- ALT PSI minus REF PSI in percentage points.
- delta_psi_sd
- Standard deviation of ΔPSI across the three transfection replicates, when reported by the source.
- delta_psi_p_value
- Two-tailed Student's t-test p-value for the REF/ALT ΔPSI comparison.
- delta_psi_fdr
- False-discovery rate for ΔPSI calculated across the PL2 library by the authors.
- splicing_call_fdr_0_05
- Derived call using the paper's ±5 percentage-point and FDR <0.05 criteria: SDV_gain_inclusion, SDV_loss_inclusion, or not_SDV_at_FDR_0.05.
- source_file
- Raw supplementary workbook from which the row was extracted.
- source_row
- Physical Excel row number in source_file.
- qc_status
- Indicates that the row was in an author post-QC analyzed sheet and passed the package's cell-line readout filter.
Quality control
The paper removed barcode constructs with barcode mutations, fewer than 85% correct MiSeq reads for the associated test sequence, absence from PL1, synthesis errors, or no PL2 reads. UMI duplicates were removed from transcript reads; barcodes with fewer than 10 RNA-seq reads were discarded. Variants were retained for ΔPSI only when at least two expressed barcodes were available for each allele in each transfection experiment and PSI was greater than zero for at least one allele. The authors' analyzed sheets retain 183/210 Var.GWAS and 121/146 Var.P variants after these filters. Package QC additionally requires a numeric REF PSI mean, ALT PSI mean, and ΔPSI for this cell line, yielding 318 rows (20 positive controls, 179 Var.GWAS, and 119 Var.P); non-significant variants are retained.
Curation notes
Vex-seq is the authors' terminology; it is categorized here as a Massively Parallel Splicing Assay because the readout is allele-dependent exon inclusion rather than generic reporter expression. This child experiment uses the same pooled library as the HEK293T child experiment but has a separate MAC-T biosample. The final table is variant-level and includes the author's source-row provenance; the raw oligo/barcode designs are in supplementary_data_7_vexseq_oligonucleotides.xlsx. Positive controls include bovine and human constructs, while the primary study target is Bos taurus. Human control coordinates in the positive-control workbook may refer to GRCh37/hg19 rather than ARS-UCD1.2. Raw FASTQ files were intentionally not packaged.