PMD Uncovers Widespread Cell-State Erasure by scRNAseq Batch Correction Methods

Single cell RNAseq (scRNAseq) batches range from technical replicates to multi-tissue atlases, thus requiring robust batch correction methods that operate effectively across this similarity spectrum. Currently, no metrics allow for full benchmarking across this spectrum, resulting in benchmarks that quantify removal of batch effects without quantifying preservation of real batch differences. Here, we address these gaps with a new statistical metric [Percent Maximum Difference (PMD)] that linearly quantifies batch similarity, and simulations generating cells from mixtures of distinct gene expression programs (cell-lineages/-types/-states). Using 690 real-world and 672 simulated integrations (7.2e6 cells total) we compared 7 batch integration approaches across the spectrum of similarity with batch-confounded gene expression. Count downsampling appeared the most robust, while others left residual batch effects or produced over-merged datasets. We further released open-source PMD and downsampling packages, with the latter capable of downsampling an organism atlas (245,389 cells) in tens of minutes on a standard computer.

DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. This process is facilitated by the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.

DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice.

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. Underlying this process is the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.

The mitochondrial genome of the grape powdery mildew pathogen Erysiphe necator is intron rich and exhibits a distinct gene organization

Powdery mildews are notorious fungal plant pathogens but only limited information exists on their genomes. Here we present the mitochondrial genome of the grape powdery mildew fungus Erysiphe necator and a high-quality mitochondrial gene annotation generated through cloning and Sanger sequencing of full-length cDNA clones. The E. necator mitochondrial genome consists of a circular DNA sequence of 188,577 bp that harbors a core set of 14 protein-coding genes that are typically present in fungal mitochondrial genomes, along with genes encoding the small and large ribosomal subunits, a ribosomal protein S3, and 25 mitochondrial-encoded transfer RNAs (mt-tRNAs). Interestingly, it also exhibits a distinct gene organization with atypical bicistronic-like expression of the nad4L/nad5 and atp6/nad3 gene pairs, and contains a large number of 70 introns, making it one of the richest in introns mitochondrial genomes among fungi. Sixty-four intronic ORFs were also found, most of which encoded homing endonucleases of the LAGLIDADG or GIY-YIG families. Further comparative analysis of five E. necator isolates revealed 203 polymorphic sites, but only five were located within exons of the core mitochondrial genes. These results provide insights into the organization of mitochondrial genomes of powdery mildews and represent valuable resources for population genetic and evolutionary studies.