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1.
Nat Biotechnol ; 2024 Jan 02.
Article in English | MEDLINE | ID: mdl-38168995

ABSTRACT

Tandem repeat (TR) variation is associated with gene expression changes and numerous rare monogenic diseases. Although long-read sequencing provides accurate full-length sequences and methylation of TRs, there is still a need for computational methods to profile TRs across the genome. Here we introduce the Tandem Repeat Genotyping Tool (TRGT) and an accompanying TR database. TRGT determines the consensus sequences and methylation levels of specified TRs from PacBio HiFi sequencing data. It also reports reads that support each repeat allele. These reads can be subsequently visualized with a companion TR visualization tool. Assessing 937,122 TRs, TRGT showed a Mendelian concordance of 98.38%, allowing a single repeat unit difference. In six samples with known repeat expansions, TRGT detected all expansions while also identifying methylation signals and mosaicism and providing finer repeat length resolution than existing methods. Additionally, we released a database with allele sequences and methylation levels for 937,122 TRs across 100 genomes.

2.
Clin Epigenetics ; 15(1): 127, 2023 08 07.
Article in English | MEDLINE | ID: mdl-37550724

ABSTRACT

BACKGROUND: As the average age of fatherhood increases worldwide, so too does the need for understanding effects of aging in male germline cells. Molecular change, including epigenomic alterations, may impact offspring. Age-associated change to DNA cytosine methylation in the cytosine-guanine (CpG) context is a hallmark of aging tissues, including sperm. Prior studies have led to accurate models that predict a man's age based on specific methylation features in the DNA of sperm, but the relationship between aging and global DNA methylation in sperm remains opaque. Further clarification requires a more complete survey of the methylome with assessment of variability within and between individuals. RESULTS: We collected sperm methylome data in a longitudinal study of ten healthy fertile men. We used whole-genome bisulfite sequencing of samples collected 10 to 18 years apart from each donor. We found that, overall, variability between donors far exceeds age-associated variation. After controlling for donor identity, we see significant age-dependent genome-wide change to the methylome. Notably, trends of change with age depend on genomic location or annotation, with contrasting signatures that correlate with gene density and proximity to centromeres and promoter regions. CONCLUSIONS: We uncovered epigenetic signatures that reflect a stable process which begins in early adulthood, progressing steadily through most of the male lifespan, and warrants consideration in any future study of the aging sperm epigenome.


Subject(s)
DNA Methylation , Epigenome , Humans , Male , Adult , Longitudinal Studies , Semen , Spermatozoa/metabolism , Aging/genetics , Cytosine/metabolism , Epigenesis, Genetic
3.
Dev Cell ; 56(16): 2381-2398.e6, 2021 08 23.
Article in English | MEDLINE | ID: mdl-34428401

ABSTRACT

Congenital abnormalities of the kidney and urinary tract are among the most common birth defects, affecting 3% of newborns. The human kidney forms around a million nephrons from a pool of nephron progenitors over a 30-week period of development. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlage, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single-cell RNA sequencing identified 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 predicted 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.


Subject(s)
Gene Expression Regulation, Developmental , Nephrons/metabolism , Transcriptome , Animals , Humans , Mice , Nephrons/cytology , Nephrons/embryology , Proteome/genetics , Proteome/metabolism , RNA-Seq , Single-Cell Analysis
4.
NAR Genom Bioinform ; 3(4): lqab115, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34988438

ABSTRACT

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.

5.
Dev Cell ; 50(1): 102-116.e6, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31265809

ABSTRACT

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.


Subject(s)
Cell Differentiation , Gene Expression Regulation, Developmental , Induced Pluripotent Stem Cells/cytology , Kidney Glomerulus/cytology , Organoids/cytology , Podocytes/cytology , Single-Cell Analysis/methods , Cells, Cultured , Female , Humans , Induced Pluripotent Stem Cells/metabolism , Kidney Glomerulus/metabolism , Organoids/metabolism , Podocytes/metabolism
6.
F1000Res ; 8: 1874, 2019.
Article in English | MEDLINE | ID: mdl-33552473

ABSTRACT

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.


Subject(s)
Software , Quality Control
8.
Dev Cell ; 45(5): 651-660.e4, 2018 06 04.
Article in English | MEDLINE | ID: mdl-29870722

ABSTRACT

Mammalian nephrons arise from a limited nephron progenitor pool through a reiterative inductive process extending over days (mouse) or weeks (human) of kidney development. Here, we present evidence that human nephron patterning reflects a time-dependent process of recruitment of mesenchymal progenitors into an epithelial nephron precursor. Progressive recruitment predicted from high-resolution image analysis and three-dimensional reconstruction of human nephrogenesis was confirmed through direct visualization and cell fate analysis of mouse kidney organ cultures. Single-cell RNA sequencing of the human nephrogenic niche provided molecular insights into these early patterning processes and predicted developmental trajectories adopted by nephron progenitor cells in forming segment-specific domains of the human nephron. The temporal-recruitment model for nephron polarity and patterning suggested by direct analysis of human kidney development provides a framework for integrating signaling pathways driving mammalian nephrogenesis.


Subject(s)
Cell Differentiation , Gene Expression Regulation, Developmental , Mesenchymal Stem Cells/cytology , Nephrons/cytology , Organogenesis/physiology , Animals , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Mesenchymal Stem Cells/metabolism , Mice , Nephrons/metabolism , Signal Transduction , Single-Cell Analysis , Time Factors
9.
J Am Soc Nephrol ; 29(3): 806-824, 2018 03.
Article in English | MEDLINE | ID: mdl-29449449

ABSTRACT

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.


Subject(s)
Kidney Cortex/embryology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Nephrons/embryology , Animals , Apoptosis Regulatory Proteins , Cell Differentiation , Cell Lineage , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Male , Mice , Myeloid Ecotropic Viral Integration Site 1 Protein/genetics , Myeloid Ecotropic Viral Integration Site 1 Protein/metabolism , Nephrons/anatomy & histology , Nephrons/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Sequence Analysis, RNA , Single-Cell Analysis , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
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