Experiment / E2438D8A1AAV-MPRA / in vivo MPRA

STAR408 scAAV MPRA of human enhancer candidates in early postnatal mouse brain

Parallel functional testing identifies enhancers active in early postnatal mouse brain

The main STAR408 screen tested 408 approximately 900 bp human candidate regulatory sequences from GWAS, LD, FBDHS, and PutEnh design groups in P7 mouse forebrain after P0 scAAV delivery. Four biological replicate DNA and RNA libraries were used to quantify enhancer activity by normalized RNA/DNA ratios and a GC-adjusted residual model; the packaged table retains the authors' 308 QC-passing amplicons.

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Organism: Mouse
Taxonomy ID: NCBITaxon:10090
Biosample: UBERON:0000955
Reference genome: GRCh38
Design focus: Region-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

Human amplicons were cloned in the 3′ UTR of an EGFP reporter driven by the Hsp1a minimal promoter, using a modified STARR-seq orientation. The library was packaged in scAAV9(2YF), injected into the left prefrontal cortex of four P0 mouse brains, and collected at P7. Viral genomic DNA served as the delivery/input control and DNase-treated total-RNA cDNA provided the reporter output; Illumina amplicon sequencing was deduplicated and aligned to GRCh38. The paper also generated one matched high-cycle RNA technical replicate (L4 RNA, 35 PCR cycles), which is retained as a count column but excluded from the four-replicate activity summaries.

Processed data

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Column dictionary · 78 definitions

amplicon_id: Unique numeric amplicon identifier from the authors' Amp_number field.
amplicon_name: Author-provided amplicon label, including number and locus/design token.
design_group: Library design group: GWAS, LD, FBDHS, or PutEnh.
chromosome: GRCh38 chromosome for the activity-table amplicon, with chr prefix.
start_grch38: GRCh38 start coordinate from the authors' activity table.
end_grch38: GRCh38 end coordinate from the authors' activity table.
length_bp: Inclusive coordinate span calculated as end_grch38 - start_grch38 + 1.
sequence_grch38: In-silico PCR amplicon sequence from supplementary file 9.
sequence_length_bp: Length in base pairs of sequence_grch38.
sequence_gc_fraction: GC fraction reported for the in-silico PCR sequence in supplementary file 9.
model_gc_fraction: Rounded GC covariate used in the authors' activity model.
previral_maxiprep_count: Raw count in the previral Maxiprep library.
previral_maxiprep_proportion: Previral Maxiprep count normalized to the total Maxiprep library count.
dna_rep_1_count: Raw viral genomic-DNA count for biological replicate L1.
dna_rep_2_count: Raw viral genomic-DNA count for biological replicate L2.
dna_rep_3_count: Raw viral genomic-DNA count for biological replicate L3.
dna_rep_4_count: Raw viral genomic-DNA count for biological replicate L4.
rna_rep_1_count: Raw reporter-RNA/cDNA count for biological replicate L1.
rna_rep_2_count: Raw reporter-RNA/cDNA count for biological replicate L2.
rna_rep_3_count: Raw reporter-RNA/cDNA count for biological replicate L3.
rna_rep_4_count: Raw reporter-RNA/cDNA count for biological replicate L4.
rna_rep_4_technical_35cycles_count: L4 reporter-RNA/cDNA count from the matched 35-PCR-cycle technical replicate; not used in summary activity.
dna_count_mean: Mean raw DNA count across L1-L4.
dna_count_sd: Standard deviation of raw DNA counts across L1-L4.
rna_count_mean: Mean raw RNA/cDNA count across L1-L4.
rna_count_sd: Standard deviation of raw RNA/cDNA counts across L1-L4.
dna_rep_1_proportion: L1 DNA count normalized to the total L1 DNA library count.
dna_rep_2_proportion: L2 DNA count normalized to the total L2 DNA library count.
dna_rep_3_proportion: L3 DNA count normalized to the total L3 DNA library count.
dna_rep_4_proportion: L4 DNA count normalized to the total L4 DNA library count.
rna_rep_1_proportion: L1 RNA/cDNA count normalized to the total L1 RNA library count.
rna_rep_2_proportion: L2 RNA/cDNA count normalized to the total L2 RNA library count.
rna_rep_3_proportion: L3 RNA/cDNA count normalized to the total L3 RNA library count.
rna_rep_4_proportion: L4 RNA/cDNA count normalized to the total L4 RNA library count.
dna_prop_mean: Mean normalized DNA proportion across L1-L4.
dna_prop_sd: Standard deviation of normalized DNA proportions across L1-L4.
rna_prop_mean: Mean normalized RNA proportion across L1-L4.
rna_prop_sd: Standard deviation of normalized RNA proportions across L1-L4.
activity_rep_1: L1 ratiometric activity, normalized RNA proportion divided by normalized DNA proportion.
activity_rep_2: L2 ratiometric activity, normalized RNA proportion divided by normalized DNA proportion.
activity_rep_3: L3 ratiometric activity, normalized RNA proportion divided by normalized DNA proportion.
activity_rep_4: L4 ratiometric activity, normalized RNA proportion divided by normalized DNA proportion.
mean_activity: Mean of L1-L4 ratiometric activities.
sd_activity: Standard deviation of L1-L4 ratiometric activities.
mean_ratio_of_means: RNA_prop_mean divided by DNA_prop_mean.
mean_ratio_replicate_activity: Mean ratiometric activity calculated from the four replicate activity ratios.
mean_ratio_sd: Standard deviation of the four replicate activity ratios.
activity_rank: Rank of mean_ratio_of_means among the QC-passing amplicons.
model_residual: Manual residual from the GC-adjusted linear activity model.
model_residual_z: Residual standardized to the fitted linear-model residual distribution.
model_p_value: One-tailed model p-value for increased RNA activity based on the standardized residual.
model_q_value_two_tailed: Empirical tail-area FDR q-value from the authors' residual analysis, reported as two-tailed.
model_fdr_lt_0_1: Author-provided boolean for the one-tailed model q-value meeting FDR<0.1.
wilcoxon_p_value: Two-tailed Wilcoxon rank-sum p-value comparing DNA and RNA proportions across replicates.
wilcoxon_conf_low: Lower confidence bound for the Wilcoxon activity comparison.
wilcoxon_conf_high: Upper confidence bound for the Wilcoxon activity comparison.
wilcoxon_bh_fdr: Benjamini-Hochberg-adjusted two-tailed Wilcoxon p-value.
wilcoxon_significance: Author descriptor of one-tailed Wilcoxon significance.
model_p_lt_0_05: Derived boolean indicating model_p_value<0.05 with positive model_residual.
ratiometric_active: Derived boolean indicating mean_ratio_replicate_activity>1.5 and mean_ratio_sd<mean_ratio_replicate_activity.
dnase_roadmap: Whether the amplicon intersects a Roadmap fetal-brain DNase hypersensitivity interval.
h3k27me3_roadmap: Whether the amplicon intersects a Roadmap H3K27me3 interval.
h3k36me3_roadmap: Whether the amplicon intersects a Roadmap H3K36me3 interval.
h3k4me1_roadmap: Whether the amplicon intersects a Roadmap H3K4me1 interval.
h3k4me3_roadmap: Whether the amplicon intersects a Roadmap H3K4me3 interval.
h3k9me3_roadmap: Whether the amplicon intersects a Roadmap H3K9me3 interval.
fetal_brain_tf_footprints_boca: Whether the amplicon intersects fetal-brain TF footprints from BOCA.
fetal_lung_tf_footprints_boca: Whether the amplicon intersects fetal-lung TF footprints from BOCA.
k562_tf_footprints_boca: Whether the amplicon intersects K562-cell TF footprints from BOCA.
atac_human_neurons_boca: Whether the amplicon intersects human-neuron ATAC-seq peaks from BOCA.
atac_human_glia_boca: Whether the amplicon intersects human-glia ATAC-seq peaks from BOCA.
conserved_element_boca: Whether the amplicon intersects a vertebrate conserved element annotation.
snp_count: Number of SNP observations associated with the amplicon in supplementary file 7.
snp_ids: Semicolon-separated unique SNP identifiers associated with the amplicon.
snp_positions_grch38: Semicolon-separated chr:position SNP coordinates on GRCh38.
allele_qc_snp_count: Number of associated SNP observations with Min_DNA_depth, Min_RNA_depth, and Minor_allele_freq_threshold all true.
allele_qc_snp_ids: Semicolon-separated SNP IDs meeting all three author allele-analysis QC flags.
qc_pass: Package QC retention flag; true for rows inherited from the authors' 308-row QC-filtered activity table.

Quality control

The authors first required at least 200 previral Maxiprep reads per amplicon, yielding 345 successfully cloned elements from the 408-element design. Downstream QC removed amplicons with fewer than 200 raw counts in any DNA sample or a mean DNA-library proportion below 2^-15 in any DNA sample, retaining 308/345 (89%) elements. The processed table uses that author-provided 308-row QC-filtered set, keeps four biological replicates, and excludes the L4 35-cycle technical RNA replicate from summary activity metrics. As validation, all 308 rows have unique IDs and finite source activity values; the packaged table reproduces the paper's 41 model p<0.05 calls, 17 model FDR<0.1 calls, 71 ratiometric calls, and 77 high-coverage/MAF-qualified SNP observations.

Curation notes

The full screen is the primary MPRA experiment; its human candidate sequences are assayed in mouse brain, so GRCh38 describes the library while the target organism and biosample describe the in vivo host. The allele-specific analysis is secondary and the paper notes limited power for moderate/small allelic effects; raw_data/supplementary_file_7_SNP_All_440.csv retains all 440 SNP observations, while the processed table aggregates the 413 observations associated with retained amplicons and the 77 observations meeting the paper's high-coverage/MAF flags. Seven retained amplicons had coordinate discrepancies between activity and in-silico PCR tables, so their unique primer-ID sequence records were used; two of those sequences are one base shorter than the activity coordinate span.

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