Biology subjects

Ussery, D. W.

Publications and source records attributed to Ussery, D. W..

5 recordsLinked to original sources

bioRxiv2023

Genomic Surveillance of SARS-CoV-2 Using Long-Range PCR Primers

Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence the [~]30,000 nucleotide SARS-CoV-2 genome that use a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with Ns inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). This study uses seven long-range PCR primers with an amplicon size of [~]4500 bp to tile across the complete SARS-CoV-2 genome. One of these regions includes the full-length S-gene by using a set of flanking primers. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias.

genomics↗

bioRxiv2019

What can we learn from over 100,000 Escherichia coli genomes?

The explosion of microbial genome sequences in public databases allows for large-scale population genomic studies of bacterial species, such as Escherichia coli. In this study, we examine and classify more than one hundred thousand E. coli and Shigella genomes. After removing outliers, a semi-automated Mash-based analysis of 10,667 assembled genomes reveals 14 distinct phylogroups. A representative genome or medoid identified for each phylogroup serves as a proxy to classify more than 95,000 unassembled genomes. This analysis shows that most sequenced E. coli genomes belong to 4 phylogroups (A, C, B1 and E2(O157)). Authenticity of the 14 phylogroups described is supported by pangenomic and phylogenetic analyses, which show differences in gene preservation between phylogroups. A phylogenetic tree constructed with 2,613 single copy core genes along with a matrix of phylogenetic profiles is used to confirm that the 14 phylogroups change at different rates of gene gain/loss/duplication. The methodology used in this work is able to identify previously uncharacterized phylogroups in E. coli species. Some of these new phylogroups harbor clonal strains that have undergone a process of genomic adaptation to the acquisition of new genomic elements related to virulence or antibiotic resistance. This is, to our knowledge, the largest E. coli genome dataset analyzed to date and provides valuable insights into the population structure of the species.

genomics↗

bioRxiv2019

Improved bacteria population structure analysis on thousands of genomes using unsupervised methods

Over ten thousand genomes of Escherichia coli are now available, and this number will continue to grow for this and other important microbial species. The first approach often used to better understand microbes is phylogenetic group analysis followed by pan-genome analysis of highly related genomes. Here, we combine sequence-based features with unsupervised clustering on up to 2,231 E. coli genomes and a total of 1,367 Clostridium difficile genomes. We show that Non-negative Matrix Factorization (NMF) can identify \"mixed\"/cryptic genomes, and can better determine inter-related genome groups and their distinguishing features (genes) relative to prior methods.

bioinformatics↗

bioRxiv2018

Decoding the Epitranscriptional Landscape from Native RNA Sequences

Sequencing of native RNA and corresponding cDNA was performed using Oxford Nanopore Technology. The % Error of Specific Bases (%ESB) was higher for native RNA than for cDNA, which enabled detection of ribonucleotide modification sites. Based on %ESB differences of the two templates, a bioinformatic tool ELIGOS was developed and applied to rRNAs of E. coli, yeast and human cells. ELIGOS captured 91%, 95%, [~]75%, respectively, of the known variety of RNA methylation sites in these rRNAs. Yeast transcriptomes from different growth conditions were also compared, which identified an association between metabolic adaptation and inferred RNA modifications. ELIGOS was further applied to human transcriptome datasets, which identified the well-known DRACH motif containing N6-methyadenine being located close to 3-untranslated regions of mRNA. Moreover, the RNA G-quadruplex motif was uncovered by ELIGOS. In summary, we have developed an experimental method coupled with bioinformatic software to uncover native RNA modifications and secondary-structures within transcripts.

genetics↗

bioRxiv2018

Rapid Sequencing of Multiple RNA Viruses in their Native Form

Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97 % identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.

genomics↗