Classification and functional characterization of regulators of intracellular STING trafficking identified by genome-wide optical pooled screening.

STING is an innate immune sensor that traffics across many cellular compartments to carry out its function of detecting cyclic di-nucleotides and triggering defense processes. Mutations in factors that regulate this process are often linked to STING-dependent human inflammatory disorders. To systematically identify factors involved in STING trafficking, we performed a genome-wide optical pooled screen and examined the impact of genetic perturbations on intracellular STING localization. Based on subcellular imaging of STING protein and trafficking markers in 45 million cells perturbed with sgRNAs, we defined 464 clusters of gene perturbations with similar cellular phenotypes. A higher-dimensional focused optical pooled screen on 262 perturbed genes which assayed 11 imaging channels identified 73 finer phenotypic clusters. In a cluster containing USE1, a protein that mediates Golgi to ER transport, we found a gene of unknown function, C19orf25. Consistent with the known role of USE1, loss of C19orf25 enhanced STING signaling. Other clusters contained subunits of the HOPS, GARP and RIC1-RGP1 complexes. We show that HOPS deficiency delayed STING degradation and consequently increased signaling. Similarly, GARP/RIC1-RGP1 loss increased STING signaling by delaying STING exit from the Golgi. Our findings demonstrate that genome-wide genotype-phenotype maps based on high-content cell imaging outperform other screening approaches, and provide a community resource for mining for factors that impact STING trafficking as well as other cellular processes observable in our dataset.

Reference genome and transcriptome informed by the sex chromosome complement of the sample increase ability to detect sex differences in gene expression from RNA-Seq data.

BACKGROUND: Human X and Y chromosomes share an evolutionary origin and, as a consequence, sequence similarity. We investigated whether the sequence homology between the X and Y chromosomes affects the alignment of RNA-Seq reads and estimates of differential expression. We tested the effects of using reference genomes and reference transcriptomes informed by the sex chromosome complement of the sample's genome on the measurements of RNA-Seq abundance and sex differences in expression. RESULTS: The default genome includes the entire human reference genome (GRCh38), including the entire sequence of the X and Y chromosomes. We created two sex chromosome complement informed reference genomes. One sex chromosome complement informed reference genome was used for samples that lacked a Y chromosome; for this reference genome version, we hard-masked the entire Y chromosome. For the other sex chromosome complement informed reference genome, to be used for samples with a Y chromosome, we hard-masked only the pseudoautosomal regions of the Y chromosome, because these regions are duplicated identically in the reference genome on the X chromosome. We analyzed the transcript abundance in the whole blood, brain cortex, breast, liver, and thyroid tissues from 20 genetic female (46, XX) and 20 genetic male (46, XY) samples. Each sample was aligned twice: once to the default reference genome and then independently aligned to a reference genome informed by the sex chromosome complement of the sample, repeated using two different read aligners, HISAT and STAR. We then quantified sex differences in gene expression using featureCounts to get the raw count estimates followed by Limma/Voom for normalization and differential expression. We additionally created sex chromosome complement informed transcriptome references for use in pseudo-alignment using Salmon. Transcript abundance was quantified twice for each sample: once to the default target transcripts and then independently to target transcripts informed by the sex chromosome complement of the sample. CONCLUSIONS: We show that regardless of the choice of the read aligner, using an alignment protocol informed by the sex chromosome complement of the sample results in higher expression estimates on the pseudoautosomal regions of the X chromosome in both genetic male and genetic female samples, as well as an increased number of unique genes being called as differentially expressed between the sexes. We additionally show that using a pseudo-alignment approach informed on the sex chromosome complement of the sample eliminates Y-linked expression in female XX samples.