Archaic introgression shaping functional genetic variation in Near Oceania
Near Oceanic populations harbour remarkable cultural, linguistic, phenotypic, and genetic diversity, yet are drastically underrepresented in human genetic studies. We reconstructed genetic sequences found in modern Near Oceanians that are derived from introgression from archaic hominins (Neanderthals and Denisovans), and characterized the functional consequences of 22,313 introgressed single nucleotide variants found at high frequency in Near Oceanians using a massively parallel reporter assay (MPRA). Across two cell lines (K562 and Jurkat), we identified 3,127 significant expression-modulating variants (emVars), and further linked 1,551 of these emVars to 1,422 target genes using gene-enhancer linkage maps. These emVars substantially augmented the set of putatively functional variants (e.g. coding and splice-disrupting variants), and genes with these putatively functional variants (especially including emVars) were significantly enriched for functions involved in several pathways including interferon gamma signalling, immune signalling, and development.Overall design: MPRA of 22,313 SNVs (44,626 oligos) plus 506 negative controls, 99 positive controls, and 104 emVar controls from published MPRAs (Tewhey et al. 2016 Cell, Xue et al. 2023 Science). Libraries were constructed following Tewhey et al. 2016 Cell, where 20 bp barcodes were added to 200 bp oligos of 170 bp of SNV and symmetric flanking sequence plus 15 bp of adapter sequence on each end, then Gibson assembled into a backbone vector. After 2x150 bp sequencing of this plasmid library on an Illumina NovaSeq for oligo-barcode matching, plasmids were digested with asiSI and GFP with a minimal TATA promoter was inserted via Gibson assembly. The resulting plasmid library was expanded, isolated, and then transfected into 5 replicates of K562 cells and 4 replicates of Jurkat cells. RNA was extracted from frozen cell pellets of each replicate, and GFP transcripts were pulled down using GFP-specific biotinylated primers and streptavidin beads. Downstream cDNA libraries were normalized for concentration based on qPCR, then amplified for 9 (K562) or 16 (Jurkat) cycles by PCR using first round PCR primers, then 6 additional PCR cycles to add Illumina adapters. This 6 additional PCR cycles to add Illumina adapters was also performed in parallel on diluted GFP-containing plasmid library. Each cDNA library was pooled with diluted GFP-containing plasmid library and 5% phiX spike-in, and sequenced at 1x75 bp on an Illumina NovaSeq or NextSeq 2000. Please see the publication associated with this project for detailed methods.Data provided: (4) PE150 sequencing runs of the oligo-barcode matching library; (4) SE75 sequencing runs of the diluted GFP-containing plasmid library per replicate per cell line; (4) SE75 sequencing runs of the cDNA per replicate per cell lineTotal: 76 sequencing runs, 5+4 replicates