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Tandem repeat (TR) variation is associated with gene expression changes and over 50 rare monogenic diseases. Recent advances in sequencing have enabled accurate, long reads that can characterize the full-length sequence and methylation profile of TRs. However, despite these advances in sequencing technology, computational methods to fully profile tandem repeats across the genome do not exist. To address this gap, we introduce tools for tandem repeat genotyping (TRGT), visualization and an accompanying TR database. TRGT accurately resolves the length and sequence composition of TR regions in the human genome. Assessing 937,122 TRs, TRGT showed a Mendelian concordance of 99.56%, allowing a single repeat unit difference. In six samples with known repeat expansions, TRGT detected all repeat expansions while also identifying methylation signals, mosaicism, and providing finer resolution of repeat length. Additionally, we release a database with allele sequences and methylation levels for 937,122 TRs across 100 genomes.

DNA methylation (DNAm) is one of the most reliable biomarkers for aging across many mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, age-dependent DNAm gain is less specified. Multiple studies have demonstrated that polycomb repressive complex 2 (PRC2) targets are enriched among the CpG sites which gain methylation with age. However, a systematic whole-genome examination of all PRC2 targets in the context of aging methylome as well as whether these associations are pan-tissue or tissue-specific is lacking. Here, by analyzing DNAm data from different assays and from multiple young and old human and mouse tissues, we found that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells gain methylation with age in all examined somatic mitotic cells. We also estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the “PRC2 clock,” defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells. In addition, we demonstrate the application of this biomarker in the evaluation of different anti-aging interventions, including dietary restriction and partial epigenetic reprogramming.

DNA cytosine methylation is an important epigenomic mark with a wide range of functions in many organisms. Whole genome bisulfite sequencing is the gold standard to interrogate cytosine methylation genome-wide. Algorithms used to map bisulfite-converted reads often encode the four-base DNA alphabet with three letters by reducing two bases to a common letter. This encoding substantially reduces the entropy of nucleotide frequencies in the resulting reference genome. Within the paradigm of read mapping by first filtering possible candidate alignments, reduced entropy in the sequence space can increase the required computing effort. We introduce another bisulfite mapping algorithm (abismal), based on the idea of encoding a four-letter DNA sequence as only two letters, one for purines and one for pyrimidines. We show that this encoding can lead to greater specificity compared to existing encodings used to map bisulfite sequencing reads. Through the two-letter encoding, the abismal software tool maps reads in less time and using less memory than most bisulfite sequencing read mapping software tools, while attaining similar accuracy. This allows in silico methylation analysis to be performed in a wider range of computing machines with limited hardware settings.

High-throughput sequencing technologies have evolved at a stellar pace for almost a decade and have greatly advanced our understanding of genome biology. In these sampling-based technologies, there is an important detail that is often overlooked in the analysis of the data and the design of the experiments, specifically that the sampled observations often do not give a representative picture of the underlying population. This has long been recognized as a problem in statistical ecology and in the broader statistics literature. In this review, we discuss the connections between these fields, methodological advances that parallel both the needs and opportunities of large-scale data analysis, and specific applications in modern biology. In the process we describe unique aspects of applying these approaches to sequencing technologies, including sequencing error, population and individual heterogeneity, and the design of experiments.

DNA cytosine methylation is an important epigenomic mark with a wide range of functions across many organisms. Whole genome bisulfite sequencing (WGBS) is the gold standard to interrogate cyto-sine methylation genome-wide. Algorithms used to map WGBS reads often encode the four-base DNA alphabet with three letters by reducing two bases to a common letter. This encoding substantially reduces the entropy of nucleotide frequencies in the resulting reference genome. Within the paradigm of read mapping by first filtering possible candidate alignments, reduced entropy of the reference can increase the required computing effort. We introduce another bisulfite mapping algorithm (abismal), based on the idea of encoding a four-letter DNA sequence as only two letters, one for purines and one for pyrimidines. We show that this encoding has greater specificity when subsequences are selected from reads for filtration. Through the two-letter encoding, the abismal software tool maps reads in less time and using less memory than most WGBS read mapping software tools, while attaining similar accuracy. This allows in silico methylation analysis to be performed in a wider range of computing machines with limited hardware settings.

SummaryCongenital abnormalities of the kidney and urinary tract are amongst the most common birth defects affecting 3% of newborns. The human kidney develops over a 30-week period in which a nephron progenitor pool gives rise to around a million nephrons. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlagen, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single cell RNA sequencing identified novel progenitor states which were spatially mapped to the nephron anatomy enabling the generation of functional gene-networks predicting interactions within and between nephron cell-types. Network mining identified known developmental disease genes and predicts new targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease, and enhance efforts to generate new kidney structures.

Quality control is an essential first step in sequencing data analysis, and software tools for quality control are deeply entrenched in standard pipelines at most sequencing centers. Although the associated computations are straightforward, in many settings the total computing effort required for quality control is appreciable and warrants optimization. We present falco, an emulation of the popular FastQC tool that runs on average three times faster while generating equivalent results. Compared to FastQC, falco also provides greater scalability for datasets with longer reads and more flexible visualization of HTML reports.

Summary The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.

This study aimed to investigate the influence of tetraconazole and malathion, both used in agricultural activities, on resistance to fluconazole, itraconazole and voriconazole in Candida parapsilosis ATCC 22019. The susceptibility to tetraconazole, malathion, fluconazole, itraconazole and voriconazole, through broth microdilution. Then, 12 independent replicates, were separated and exposed to four treatment groups, each one containing three replicates: G1: tetraconazole; G2: malathion; G3: fluconazole (positive control); G4: negative control. Replicates from G1, G2 and G3, were exposed to weekly increasing concentrations of tetraconazole, malathion and fluconazole, respectively, ranging from MIC/2 to 32 × MIC, throughout 7 weeks. The exposure to tetraconazole, but not malathion, decreased susceptibility to clinical azoles, especially fluconazole. The tetraconazole-induced fluconazole resistance is partially mediated by the increased activity of ATP-dependent efflux pumps, considering the increase in antifungal susceptibility after the addition of the efflux pump inhibitor, promethazine, and the increase in rhodamine 6G efflux and CDR gene expression in the G1 replicates. Moreover, MDR expression was only detected in G1 and G3 replicates, suggesting that MDR pumps are also involved in tetraconazole-induced fluconazole resistance. It is noteworthy that tetraconazole and fluconazole-treated replicates behaved similarly, therefore, resistance to azoles of clinical use may be a consequence of using azoles in farming activities.

Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2(+) nephron progenitor cells (NPCs) and Foxd1(+) interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2(+) NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2(+) NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.