Biology subjects

Close, D.

Publications and source records attributed to Close, D..

5 recordsLinked to original sources

bioRxiv2026

Multisite Evaluation of an Amplification-based Nanopore Sequencing Solution to Analyze Challenging Clinically Relevant Variants in Genes Associated with Hereditary Diseases

PurposeCarrier screening for hereditary conditions is challenged by genes with complex genomic architecture, where short-read sequencing can fail to detect clinically relevant variants. This study evaluated a unified, amplification-based nanopore sequencing workflow across multiple laboratories for comprehensive analysis of such loci. MethodsA modular long-read sequencing assay was evaluated across five laboratories using targeted PCR enrichment, Oxford Nanopore sequencing, and automated variant analysis. The workflow interrogated genes associated with spinal muscular atrophy, thalassemia, cystic fibrosis, fragile X syndrome, congenital adrenal hyperplasia, Gaucher disease, and hemophilia A. Performance was assessed against orthogonal methods for single nucleotide variants (SNVs), indels, copy-number variants, repeat expansions, and structural rearrangements. ResultsAcross 882 unique samples (1,266 tests), overall agreement with comparator methods exceeded 96% for variant-level detection and 97% for genotype status classification. Long-read sequencing enabled phasing of paralogous loci, integrated sizing and interruption analysis for FMR1 repeats, and simultaneous detection of SNVs and structural variants in globin loci and CYP21A2-TNXB region, reducing reliance on multiple workflows. ConclusionThis multisite evaluation suggests that targeted long-read sequencing can consolidate complex variant detection into a single workflow, improving analytical completeness and operational efficiency for carrier screening.

genetics↗

bioRxiv2021

Dynamics of lipids in the yeast lipid droplets

The physical properties and chemical composition of lipid droplets inside cells of the yeast Cryptococcus curvatus were investigated using quasi-elastic neutron scattering and mass spectrometry with complementary surface modeling using 3D microscopy. With temperature decrease from 310 to 280 K, their phase remained fluid, i.e., the droplets remained in the physiological state, unlike synthetic lipid membranes that transition to a gel phase. The lipid dynamics in the droplet was described by a model implying diffusion of the lipid and its hydrocarbon chains. The diffusion coefficient of the lipid chains (274x10-3 [A]2/ps at 310 K) was much higher than that observed in a synthetic lipid membrane because of the larger volume (up to 12 [A]) for the local dynamics in the droplet. These physical properties were correlated with the types of lipids composing the droplet. Based on that, the lipid packing and resulting energetic value of the yeast droplets are discussed in relation to their usefulness as biofuels.

biophysics↗

bioRxiv2020

Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Computational analyses suggested that this bias is due to the action of restriction-modification systems whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that the intensity of recombination is uniform across the genome without concentration into hotspots. Unexpectedly, our results revealed that large species-level genetic distance did not affect key recombination parameters. Our study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

microbiology↗

bioRxiv2018

Algorithmic improvements for discovery of germline copy number variants in next-generation sequencing data

Copy number variants (CNVs) play a significant role in human heredity and disease, however sensitive and specific characterization of CNVs from NGS data has remained challenging. Detection is especially problematic for hybridization-capture data in which read counts are the sole source of copy number information. We describe two algorithmic adaptations that improve CNV detection accuracy in a Hidden Markov Model (HMM) context. First, we present a method for com puting target- and copy number state-specific emission distributions. Second, we demonstrate that the Pointwise Maximum a posteriori (PMAP) HMM decoding procedure yields improved sensitivity for small CNV calls compared to the more common Viterbi HMM decoder. We develop a prototype implementation, called Cobalt, and compare it to other CNV detection tools using sets of simulated and previously detected CNVs with sizes spanning a single exon up to a full chromosome. In both the simulation and previously detected CNV studies Cobalt shows similar sensitivity but significantly improved positive predictive value (PPV) compared to other callers. Overall sensitivity is 80%-90% for deletion CNVs spanning 1-4 targets and 90%-100% for larger deletion events, while sensitivity is somewhat lower for small duplication CNVs. Cobalt demonstrates significantly improved positive predictive value (PPV) compared to other callers with similar sensitivity, typically making 5X fewer total calls overall.

bioinformatics↗

bioRxiv2018

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis

Metabolic pathways are frequently transferred between bacterial strains in the environment through horizontal gene transfer (HGT), yet laboratory engineering to introduce new metabolic pathways often fails. Successful use of a pathway requires co-evolution of both pathway and host, and these interactions may be disrupted upon transfer to a new host. Here we show that two different pathways for catabolism of coumarate failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly-acquired pathways and the modifications required to overcome these limitations. Both pathways required mutations to the host to enable effective growth with coumarate, but the necessary mutations differed depending on the chemistry and intermediates of the pathways. In one case, an intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid mutations to IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is already resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. These discoveries will aid in our understanding of HGT and ability to predictably engineer metabolism.\n\nThis manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

microbiology↗