Steven Flygare

VAAST 2.0: Improved Variant Classification and Disease-Gene Identification Using a Conservation-Controlled Amino Acid Substitution Matrix

The need for improved algorithmic support for variant prioritization and disease‐gene identification in personal genomes data is widely acknowledged. We previously presented the Variant Annotation, Analysis, and Search Tool (VAAST), which employs an aggregative variant association test that combines both amino acid substitution (AAS) and allele frequencies. Here we describe and benchmark VAAST 2.0, which uses a novel conservation‐controlled AAS matrix (CASM), to incorporate information about phylogenetic conservation. We show that the CASM approach improves VAASTs variant prioritization accuracy compared to its previous implementation, and compared to SIFT, PolyPhen‐2, and MutationTaster. We also show that VAAST 2.0 outperforms KBAC, WSS, SKAT, and variable threshold (VT) using published case‐control datasets for Crohn disease (NOD2), hypertriglyceridemia (LPL), and breast cancer (CHEK2). VAAST 2.0 also improves search accuracy on simulated datasets across a wide range of allele frequencies, population‐attributable disease risks, and allelic heterogeneity, factors that compromise the accuracies of other aggregative variant association tests. We also demonstrate that, although most aggregative variant association tests are designed for common genetic diseases, these tests can be easily adopted as rare Mendelian disease‐gene finders with a simple ranking‐by‐statistical‐significance protocol, and the performance compares very favorably to state‐of‐art filtering approaches. The latter, despite their popularity, have suboptimal performance especially with the increasing case sample size.

Muscle stem cells contribute to myofibres in sedentary adult mice

Skeletal muscle is essential for mobility, stability, and whole body metabolism, and muscle loss, for instance during sarcopenia, has profound consequences. Satellite cells (muscle stem cells) have been hypothesized, but not yet demonstrated, to contribute to muscle homeostasis and a decline in their contribution to myofiber homeostasis to play a part in sarcopenia. To test their role in muscle maintenance, we genetically labeled and ablated satellite cells in adult sedentary mice. We demonstrate via genetic lineage experiments that even in the absence of injury, satellite cells contribute to myofibers in all adult muscles, although the extent and timing differs. However, genetic ablation experiments showed that satellite cells are not globally required to maintain myofiber cross-sectional area of uninjured adult muscle.

Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel

ABSTRACT Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patients symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n = 42) and unselected (n = 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information.

Transiently Active Wnt/β-Catenin Signaling Is Not Required but Must Be Silenced for Stem Cell Function during Muscle Regeneration

Summary Adult muscle’s exceptional capacity for regeneration is mediated by muscle stem cells, termed satellite cells. As with many stem cells, Wnt/β-catenin signaling has been proposed to be critical in satellite cells during regeneration. Using new genetic reagents, we explicitly test in vivo whether Wnt/β-catenin signaling is necessary and sufficient within satellite cells and their derivatives for regeneration. We find that signaling is transiently active in transit-amplifying myoblasts, but is not required for regeneration or satellite cell self-renewal. Instead, downregulation of transiently activated β-catenin is important to limit the regenerative response, as continuous regeneration is deleterious. Wnt/β-catenin activation in adult satellite cells may simply be a vestige of their developmental lineage, in which β-catenin signaling is critical for fetal myogenesis. In the adult, surprisingly, we show that it is not activation but rather silencing of Wnt/β-catenin signaling that is important for muscle regeneration.

Using VAAST to Identify Disease‐Associated Variants in Next‐Generation Sequencing Data

The VAAST pipeline is specifically designed to identify disease‐associated alleles in next‐generation sequencing data. In the protocols presented in this paper, we outline the best practices for variant prioritization using VAAST. Examples and test data are provided for case‐control, small pedigree, and large pedigree analyses. These protocols will teach users the fundamentals of VAAST, VAAST 2.0, and pVAAST analyses. Curr. Protoc. Hum. Genet. 81:6.14.1‐6.14.25.

Viral pathogen detection by metagenomics and pan-viral group polymerase chain reaction in children with pneumonia lacking identifiable etiology

Summary Two broad-spectrum pathogen detection methods, next-generation sequencing and pan-viral group polymerase chain reaction, detected previously missed, putative pathogens in 34% of children hospitalized with community-acquired pneumonia with no identified etiology.

Human Bocavirus Capsid Messenger RNA Detection in Children With Pneumonia

Summary Human bocavirus messenger RNA detection in nasopharyngeal specimens from children with community-acquired pneumonia (CAP) but not in asymptomatic children undergoing elective outpatient surgery supports the pathogenic role for this virus in CAP and may be a more specific target for diagnostic testing.

ImagePlane: An Automated Image Analysis Pipeline for High-Throughput Screens Using the Planarian Schmidtea mediterranea

ImagePlane is a modular pipeline for automated, high-throughput image analysis and information extraction. Designed to support planarian research, ImagePlane offers a self-parameterizing adaptive thresholding algorithm; an algorithm that can automatically segment animals into anterior-posterior/left-right quadrants for automated identification of region-specific differences in gene and protein expression; and a novel algorithm for quantification of morphology of animals, independent of their orientations and sizes. ImagePlane also provides methods for automatic report generation, and its outputs can be easily imported into third-party tools such as R and Excel. Here we demonstrate the pipelines utility for identification of genes involved in stem cell proliferation in the planarian Schmidtea mediterranea. Although designed to support planarian studies, ImagePlane will prove useful for cell-based studies as well.

771Can Human Bocavirus mRNA Detection Differentiate Acute Infection from Viral Shedding

from Viral Shedding? Robert Schlaberg, MD, MPH; Krow Ampofo, MD; Keith Tardif, PhD; Chris Stockmann, MSc; Keith Simmon, MS; Weston Hymas, MS, MB(ASCP); Steven Flygare, MS; Brett Kennedy, PhD; Anne J. Blaschke, MD, PhD; Karen Eilbeck, PhD; Mark Yandell, PhD; Anna M. Bramley, MPH; Seema Jain, MD, MPH; Andrew Pavia, MD, FIDSA, FSHEA; Department of Pathology, University of Utah, Salt Lake City, UT; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT; Department of Pediatrics, Division of Pediatric Infectious Diseases, University of Utah School of Medicine, Salt Lake City, UT; Department of Biomedical Informatics, University of Utah, Salt Lake City, UT; Department of Human Genetics, University of Utah, Salt Lake City, UT; Centers for Disease Control and Prevention, Atlanta, GA; Centers for Disease Control and Prevention, Atlanta, GA