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1. Development and Validation of a Parsimonious Tuberculosis Gene Signature Using the digital NanoString nCounter Platform

   https://doi.org/10.1093/cid/ciac010  

   Kaipilyawar, Vaishnavi ; Zhao, Yue ; Wang, Xutao ; Joseph, Noyal M. ; Knudsen, Selby ; Prakash Babu, Senbagavalli ; Muthaiah, Muthuraj ; Hochberg, Natasha S. ; Sarkar, Sonali ; Horsburgh, Jr, Charles R. ; et al ( January 2022 , Clinical Infectious Diseases)

   Blood-based biomarkers for diagnosing active tuberculosis (TB), monitoring treatment response, and predicting risk of progression to TB disease have been reported. However, validation of the biomarkers across multiple independent cohorts is scarce. A robust platform to validate TB biomarkers in different populations with clinical end points is essential to the development of a point-of-care clinical test. NanoString nCounter technology is an amplification-free digital detection platform that directly measures mRNA transcripts with high specificity. Here, we determined whether NanoString could serve as a platform for extensive validation of candidate TB biomarkers.

   The NanoString platform was used for performance evaluation of existing TB gene signatures in a cohort in which signatures were previously evaluated on an RNA-seq dataset. A NanoString codeset that probes 107 genes comprising 12 TB signatures and 6 housekeeping genes (NS-TB107) was developed and applied to total RNA derived from whole blood samples of TB patients and individuals with latent TB infection (LTBI) from South India. The TBSignatureProfiler tool was used to score samples for each signature. An ensemble of machine learning algorithms was used to derive a parsimonious biomarker.

   Gene signatures present in NS-TB107 had statistically significant discriminative power for segregating TB from LTBI. Further analysis of the data yielded a NanoString 6-gene set (NANO6) that when tested on 10 published datasets was highly diagnostic for active TB.

   The NanoString nCounter system provides a robust platform for validating existing TB biomarkers and deriving a parsimonious gene signature with enhanced diagnostic performance.

    

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2. A Far‐Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug‐Susceptibility Testing

   https://doi.org/10.1002/ange.202213563  

   Banahene, Nicholas ; Gepford, Dana M. ; Biegas, Kyle J. ; Swanson, Daniel H. ; Hsu, Yen‐Pang ; Murphy, Brennan A. ; Taylor, Zachary E. ; Lepori, Irene ; Siegrist, M. Sloan ; Obregón‐Henao, Andrés ; et al ( December 2022 , Angewandte Chemie)

   Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green‐emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose‐based fluorogenic probe featuring a molecular rotor turn‐on fluorophore with bright far‐red emission (RMR‐Tre). RMR‐Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no‐wash, low‐background fluorescence detection of live mycobacteria. Aided by the red‐shifted molecular rotor fluorophore, RMR‐Tre exhibited up to a 100‐fold enhancement inM. tuberculosislabeling compared to existing fluorogenic trehalose probes. We show that RMR‐Tre reports onM. tuberculosisdrug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.

    

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3. A Far‐Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug‐Susceptibility Testing

   https://doi.org/10.1002/anie.202213563  

   Banahene, Nicholas ; Gepford, Dana M. ; Biegas, Kyle J. ; Swanson, Daniel H. ; Hsu, Yen‐Pang ; Murphy, Brennan A. ; Taylor, Zachary E. ; Lepori, Irene ; Siegrist, M. Sloan ; Obregón‐Henao, Andrés ; et al ( December 2022 , Angewandte Chemie International Edition)

   Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green‐emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose‐based fluorogenic probe featuring a molecular rotor turn‐on fluorophore with bright far‐red emission (RMR‐Tre). RMR‐Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no‐wash, low‐background fluorescence detection of live mycobacteria. Aided by the red‐shifted molecular rotor fluorophore, RMR‐Tre exhibited up to a 100‐fold enhancement inM. tuberculosislabeling compared to existing fluorogenic trehalose probes. We show that RMR‐Tre reports onM. tuberculosisdrug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.

    

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4. P ool P arty 2: An integrated pipeline for analysing pooled or indexed low‐coverage whole‐genome sequencing data to discover the genetic basis of diversity

   https://doi.org/10.1111/1755-0998.13888  

   Willis, Stuart ; Micheletti, Steven ; Andrews, Kimberly R. ; Narum, Shawn ( November 2023 , Molecular Ecology Resources)

   Whole‐genome sequencing data allow survey of variation from across the genome, reducing the constraint of balancing genome sub‐sampling with estimating recombination rates and linkage between sampled markers and target loci. As sequencing costs decrease, low‐coverage whole‐genome sequencing of pooled or indexed‐individual samples is commonly utilized to identify loci associated with phenotypes or environmental axes in non‐model organisms. There are, however, relatively few publicly available bioinformatic pipelines designed explicitly to analyse these types of data, and fewer still that process the raw sequencing data, provide useful metrics of quality control and then execute analyses. Here, we present an updated version of a bioinformatics pipeline calledPoolParty2that can effectively handle either pooled or indexed DNA samples and includes new features to improve computational efficiency. Using simulated data, we demonstrate the ability of our pipeline to recover segregating variants, estimate their allele frequencies accurately, and identify genomic regions harbouring loci under selection. Based on the simulated data set, we benchmark the efficacy of our pipeline with another bioinformatic suite,angsd, and illustrate the compatibility and complementarity of these suites usingangsdto generate genotype likelihoods as input for identifying linkage outlier regions using alignment files and variants provided byPoolParty2. Finally, we apply our updated pipeline to an empirical dataset of low‐coverage whole genomic data from population samples of Columbia River steelhead trout (Oncorhynchus mykiss), results from which demonstrate the genomic impacts of decades of artificial selection in a prominent hatchery stock. Thus, we not only demonstrate the utility ofPoolParty2for genomic studies that combine sequencing data from multiple individuals, but also illustrate how it compliments other bioinformatics resources such asangsd.

    

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5. Intraspecific DNA contamination distorts subtle population structure in a marine fish: Decontamination of herring samples before restriction‐site associated sequencing and its effects on population genetic statistics

   https://doi.org/10.1111/1755-0998.12978  

   Petrou, Eleni L. ; Drinan, Daniel P. ; Kopperl, Robert ; Lepofsky, Dana ; Yang, Dongya ; Moss, Madonna L. ; Hauser, Lorenz ( June 2019 , Molecular Ecology Resources)

   Wild specimens are often collected in challenging field conditions, where samples may be contaminated with the DNA of conspecific individuals. This contamination can result in false genotype calls, which are difficult to detect, but may also cause inaccurate estimates of heterozygosity, allele frequencies and genetic differentiation. Marine broadcast spawners are especially problematic, because population genetic differentiation is low and samples are often collected in bulk and sometimes from active spawning aggregations. Here, we used contaminated and clean Pacific herring (Clupea pallasi) samples to test (a) the efficacy of bleach decontamination, (b) the effect of decontamination on RAD genotypes and (c) the consequences of contaminated samples on population genetic analyses. We collected fin tissue samples from actively spawning (and thus contaminated) wild herring and nonspawning (uncontaminated) herring. Samples were soaked for 10 min in bleach or left untreated, and extracted DNA was used to prepare DNA libraries using a restriction site‐associated DNA (RAD) approach. Our results demonstrate that intraspecific DNA contamination affects patterns of individual and population variability, causes an excess of heterozygotes and biases estimates of population structure. Bleach decontamination was effective at removing intraspecific DNA contamination and compatible with RAD sequencing, producing high‐quality sequences, reproducible genotypes and low levels of missing data. Although sperm contamination may be specific to broadcast spawners, intraspecific contamination of samples may be common and difficult to detect from high‐throughput sequencing data and can impact downstream analyses.

    

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