Biology subjects

Kovar, L.

Publications and source records attributed to Kovar, L..

3 recordsLinked to original sources

bioRxiv2022

Genomic deletions and rearrangements in monkeypox virus from the 2022 outbreak, USA

Genomic surveillance of monkeypox virus (MPXV) during the 2022 outbreak has been mainly focused on single nucleotide polymorphism (SNP) changes. DNA viruses, including MPXV, have a lower SNP mutation rate than RNA viruses due to higher fidelity replication machinery. We identified a large genomic rearrangement in a MPXV sequence from a 2022 case in the state of Minnesota (MN), USA, from an abnormal, uneven MPXV read mapping coverage profile in whole-genome sequencing (WGS) data. We further screened WGS data of 206 U.S. MPXV samples and found seven (3.4 percent) sequenced genomes contained similar abnormal read coverage profiles that suggested putative large deletions or genomic rearrangements. Here, we present three MPXV genomes containing deletions ranging from 2.3 to 15 kb and four genomes containing more complex rearrangements. Five genomic changes were each only seen in one sample, but two sequences from linked cases shared an identical 2.3 kb deletion in the 3 terminal region. All samples were positive using VAC1 and Clade II (formerly West African)-specific MPXV diagnostic tests; however, large deletions and genomic rearrangements like the ones reported here have the potential to result in viruses in which the target of a PCR diagnostic test is deleted. The emergence of genomic rearrangements during the outbreak may have public health implications and highlight the importance of continued genomic surveillance.

genomics↗

bioRxiv2021

Plant-derived insulator-like sequences for control of transgene expression

Stable and consistent transgene expression is necessary to advance plant biotechnology. Stable expression can be achieved by incorporating enhancer-blocking insulators, which are cis-regulatory elements that reduce enhancer interference in gene expression, into transgene constructs. Sufficient insulators for plant use are not available, and their discovery has remained elusive. In this work, we computationally mined the compact genome of Utricularia gibba for insulator sequences and identified short (<1 kb) sequences with potential insulator activity. Based on in vivo tests, three of these effectively mitigate the ectopic transgene expression caused by the Cauliflower Mosaic Virus 35S promoter and do so better than previously reported plant insulators. However, all sequences with apparent insulator activity also decrease the effectiveness of the CaMV 35S promoter, and thus may be more accurately classified as silencers. However, since the insulator effect is proportionately much higher than the silencing effect, these sequences are still useful for plant transformation.

plant biology↗

bioRxiv2020

Comparing DNA Extraction and 16s Amplification Methods for Plant-Associated Bacterial Communities

Plant-associated microbes play important roles in global ecology and agriculture. The most common method to profile these microbial communities is amplicon sequencing of the bacterial 16s rRNA gene. Both the DNA extraction and PCR amplification steps of this process are subject to bias, especially since the latter requires some way to exclude DNA from plant organelles, which would otherwise dominate the sample. We compared several common DNA extraction kits and 16s rRNA amplification protocols to determine the relative biases of each and to make recommendations for plant microbial researchers. For DNA extraction, we found that, as expected, kits optimized for soil were the best for soil, though each still included a distinct "fingerprint" of its own biases. Plant samples were less clear, with different species having different "best" options. For 16s amplification, we find that using peptide nucleic acid (PNA) clamps provides the least taxonomic distortion, while chloroplast-discriminating primers are easy and inexpensive but present significant bias in the results. We do not recommend blocking oligos, as they involved a more complex protocol and showed significant taxonomic bias in the results. Further methods development will hopefully result in protocols that are even more reliable and less biased.

microbiology↗