Assessment of in vivo chemical mutagenesis by long-read sequencing.

Evaluating the mutagenic properties of chemicals is crucial for understanding their potential cancer risks. Recent Illumina-based error-corrected sequencing techniques have enabled the direct detection of mutations induced de novo by mutagens. However, as the Illumina platform lacks intrinsic error-correction capabilities, complex library preparations and bioinformatic processes are necessary to identify these rare mutations. In this study, we evaluated whether long-read PacBio-based HiFi sequencing (HiFi seq), which has integrated error-correction, can detect de novo mutations induced by mutagens in C57BL/6 mouse tissues. Using HiFi seq, dose-dependent increases in mutation frequencies were found in tissues from mice exposed to 7,12-dimethylbenz[a]anthracene, procarbazine, and N-propyl-N-nitrosourea. Furthermore, the mutational signatures derived from these exposures were consistent with those previously reported for these mutagens. This study demonstrates that HiFi seq can complement established mutation detection assays to facilitate the identification of hazardous compounds.

Complete genome sequence of cephalosporin and tetracycline-resistant Citrobacter freundii CF51 isolate from a patient with urinary tract infection.

We report here the complete genome sequence of cephalosporin (cefazolin and cephalotin) and tetracycline-resistant Citrobacter freundii strain CF51, isolated from a patient with a urinary tract infection. The genome comprises a 5.0-Mb chromosome, along with an 87.2-kb plasmid and a 23.9-kb plasmid.

Whole-Genome Sequence Analysis of Antibiotic Resistance, Virulence, and Plasmid Dynamics in Multidrug-Resistant E. coli Isolates from Imported Shrimp.

We analyzed antimicrobial resistance and virulence traits in multidrug-resistant (MDR) E. coli isolates obtained from imported shrimp using whole-genome sequences (WGSs). Antibiotic resistance profiles were determined phenotypically. WGSs identified key characteristics, including their multilocus sequence type (MLST), serotype, virulence factors, antibiotic resistance genes, and mobile elements. Most of the isolates exhibited resistance to gentamicin, streptomycin, ampicillin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. Multilocus sequence type (MLST), serotype, average nucleotide identity (ANI), and pangenome analysis showed high genomic similarity among isolates, except for EC15 and ECV01. The EC119 plasmid contained a variety of efflux pump genes, including those encoding the acid resistance transcriptional activators (gadE, gadW, and gadX), resistance-nodulation-division-type efflux pumps (mdtE and mdtF), and a metabolite, H1 symporter (MHS) family major facilitator superfamily transporter (MNZ41_23075). Virulence genes displayed diversity, particularly EC15, whose plasmids carried genes for adherence (faeA and faeC-I), invasion (ipaH and virB), and capsule (caf1A and caf1M). This comprehensive analysis illuminates antimicrobial resistance, virulence, and plasmid dynamics in E. coli from imported shrimp and has profound implications for public health, emphasizing the need for continued surveillance and research into the evolution of these important bacterial pathogens.

Unbiased whole genome detection of ultrarare off-target mutations in genome-edited cell populations by HiFi sequencing.

DNA base editors (BEs) composed of a nuclease-deficient Cas9 fused to a DNA-modifying enzyme can achieve on-target mutagenesis without creating double-strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease-proficient Cas9 systems, which do rely on the creation of DSBs to achieve on-target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off-target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10-8 mutations per base pair. In this proof-of-principle study, we evaluated whether this technique could also detect the on- and off-target mutations generated by a cytosine-to-thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on-target mutant allele fractions ranging from ~7% to ~63%, depending on the single-guide RNA (sgRNA) used, while no on-target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3-fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of C:G>T:A substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on- and off-target mutations in cell populations that have undergone genome editing.

Draft Genome Sequences of 14 Fluoroquinolone-Resistant Escherichia coli Isolates from Imported Shrimp.

We report the draft genome sequences of 14 fluoroquinolone-resistant Escherichia coli strains that were isolated from imported shrimp. All isolates contained multiple point mutations in the quinolone resistance-determining regions (QRDRs) and non-QRDRs of gyrA, parC, and parE genes. The data improve the understanding of fluoroquinolone resistance and indicate resistance mechanisms.

Evaluation of the mutagenic effects of Molnupiravir and N4-hydroxycytidine in bacterial and mammalian cells by HiFi sequencing.

Molnupiravir (MOV) is used to treat COVID-19. In cells, MOV is converted to the ribonucleoside analog N4-hydroxycytidine (NHC) and incorporated into the SARS-CoV-2 RNA genome during its replication, resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS-CoV-2 propagation. Although safety assessments by many regulatory agencies across the world have concluded that the genotoxic risks associated with the clinical use of MOV are low, concerns remain that it could induce DNA mutations in patients, particularly because numerous in vitro studies have shown that NHC is a DNA mutagen. In this study, we used HiFi sequencing, a technique that can detect ultralow-frequency substitution mutations in whole genomes, to evaluate the mutagenic effects of MOV in E. coli and of MOV and NHC in mouse lymphoma L5178Y cells and human lymphoblastoid TK6 cells. In all models, exposure to these compounds increased genome-wide mutation frequencies in a dose-dependent manner, and these increases were mainly composed of A:T → G:C transitions. The NHC exposure concentrations used for mammalian cells were comparable to those observed in the plasma of humans who received clinical doses of MOV.

Genome-wide detection of ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms by PacBio sequencing.

Many conventional genetic toxicology assays require specialized cell cultures or animals and can only detect mutations that inactivate the function of a reporter gene. These limitations make such assays incompatible with many toxicological models but could be overcome by the development of techniques capable of directly detecting genome-wide somatic mutations through DNA sequencing. PacBio sequencing can generate almost error-free consensus reads by repeatedly inspecting both DNA strands from circularized molecules (a method known as PacBio HiFi). In this study, we show that PacBio HiFi can detect genome-wide ultralow-frequency substitution mutations in cultures of mouse lymphoma L5178Y cells and Caenorhabditis elegans worms. The mutation frequencies (MFs) of unexposed samples in both models were ~1 × 10-7 mutations per base pair. Compared to these controls, PacBio HiFi detected MF increases of 23-fold in cultures of L5178Y cells exposed to 5 mM ethyl methanosulfonate (EMS) for 4 h, and 5-, 12-, and 29-fold in cultures of C. elegans worms exposed to 12.5, 25, and 50 mM EMS for 4 h, respectively. In both models, the mutation spectra of controls were diverse, while those derived from EMS-exposed samples were dominated by C:G → T:A transitions. To validate these results, clone sequencing analyses were performed on the same cultures of L5178Y cells. The results obtained by clone sequencing and PacBio HiFi were almost identical. Our results suggest that PacBio sequencing could be used for the detection, quantitation, and characterization of mutations in any DNA-containing sample, including those that are not compatible with conventional mutation detection approaches.

PacBio sequencing detects genome-wide ultra-low-frequency substitution mutations resulting from exposure to chemical mutagens.

Genetic toxicology uses several assays to identity mutagens and protects the public. Most of these assays, however, rely on reporter genes, can only measure mutation indirectly based on phenotype, and often require specific cell lines or animal models-features that impede their integration with existing and emerging toxicological models, such as organoids. In this study, we show that PacBio Single-Molecule, Real-Time (PB SMRT) sequencing identified substitution mutations caused by chemical mutagens in Escherichia coli by generating nearly error-free consensus reads after repeatedly inspecting both strands of circular DNA molecules. Using DNA from E. coli exposed to ethyl methanosulfonate (EMS) or N-ethyl-N-nitrosourea (ENU), PB SMRT sequencing detected mutation frequencies (MFs) and spectra comparable to those obtained by clone-sequencing from the same exposures. The optimized background MF of PB SMRT sequencing was ≤ 1 × 10-7 mutations per base pair (mut/bp).