HiFi long-read amplicon sequencing for full-spectrum variants of human mtDNA.

BACKGROUND: Mitochondrial diseases (MDs) can be caused by single nucleotide variants (SNVs) and structural variants (SVs) in the mitochondrial genome (mtDNA). Presently, identifying deletions in small to medium-sized fragments and accurately detecting low-percentage variants remains challenging due to the limitations of next-generation sequencing (NGS). METHODS: In this study, we integrated targeted long-range polymerase chain reaction (LR-PCR) and PacBio HiFi sequencing to analyze 34 participants, including 28 patients and 6 controls. Of these, 17 samples were subjected to both targeted LR-PCR and to compare the mtDNA variant detection efficacy. RESULTS: Among the 28 patients tested by long-read sequencing (LRS), 2 patients were found positive for the m.3243 A > G hotspot variant, and 20 patients exhibited single or multiple deletion variants with a proportion exceeding 4%. Comparison between the results of LRS and NGS revealed that both methods exhibited similar efficacy in detecting SNVs exceeding 5%. However, LRS outperformed NGS in detecting SNVs with a ratio below 5%. As for SVs, LRS identified single or multiple deletions in 13 out of 17 cases, whereas NGS only detected single deletions in 8 cases. Furthermore, deletions identified by LRS were validated by Sanger sequencing and quantified in single muscle fibers using real-time PCR. Notably, LRS also effectively and accurately identified secondary mtDNA deletions in idiopathic inflammatory myopathies (IIMs). CONCLUSIONS: LRS outperforms NGS in detecting various types of SNVs and SVs in mtDNA, including those with low frequencies. Our research is a significant advancement in medical comprehension and will provide profound insights into genetics.

Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants.

BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

De novo assembly of the complete mitochondrial genome of pepino (Solanum muricatum) using PacBio HiFi sequencing: insights into structure, phylogenetic implications, and RNA editing.

BACKGROUND: Solanum muricatum is an emerging horticultural fruit crop with rich nutritional and antioxidant properties. Although the chromosome-scale genome of this species has been sequenced, its mitochondrial genome sequence has not been reported to date. RESULTS: PacBio HiFi sequencing was used to assemble the circular mitogenome of S. muricatum, which was 433,466 bp in length. In total, 38 protein-coding, 19 tRNA, and 3 rRNA genes were annotated. The reticulate mitochondrial conformations with multiple junctions were verified by polymerase chain reaction, and codon usage, sequence repeats, and gene migration from chloroplast to mitochondrial genome were determined. A collinearity analysis of eight Solanum mitogenomes revealed high structural variability. Overall, 585 RNA editing sites in protein coding genes were identified based on RNA-seq data. Among them, mttB was the most frequently edited (52 times), followed by ccmB (46 times). A phylogenetic analysis based on the S. muricatum mitogenome and those of 39 other taxa (including 25 Solanaceae species) revealed the evolutionary and taxonomic status of S. muricatum. CONCLUSIONS: We provide the first report of the assembled and annotated S. muricatum mitogenome. This information will help to lay the groundwork for future research on the evolutionary biology of Solanaceae species. Furthermore, the results will assist the development of molecular breeding strategies for S. muricatum based on the most beneficial agronomic traits of this species.

Long-read genome sequencing reveals a novel intronic retroelement insertion in NR5A1 associated with 46,XY differences of sexual development.

Despite significant advancements in rare genetic disease diagnostics, many patients with rare genetic disease remain without a molecular diagnosis. Novel tools and methods are needed to improve the detection of disease-associated variants and understand the genetic basis of many rare diseases. Long-read genome sequencing provides improved sequencing in highly repetitive, homologous, and low-complexity regions, and improved assessment of structural variation and complex genomic rearrangements compared to short-read genome sequencing. As such, it is a promising method to explore overlooked genetic variants in rare diseases with a high suspicion of a genetic basis. We therefore applied PacBio HiFi sequencing in a large multi-generational family presenting with autosomal dominant 46,XY differences of sexual development (DSD), for whom extensive molecular testing over multiple decades had failed to identify a molecular diagnosis. This revealed a rare SINE-VNTR-Alu retroelement insertion in intron 4 of NR5A1, a gene in which loss-of-function variants are an established cause of 46,XY DSD. The insertion segregated among affected family members and was associated with loss-of-expression of alleles in cis, demonstrating a functional impact on NR5A1. This case highlights the power of long-read genome sequencing to detect genomic variants that have previously been intractable to detection by standard short-read genomic testing.

Genome assembly of Hibiscus sabdariffa L. provides insights into metabolisms of medicinal natural products.

Hibiscus sabdariffa L. is a widely cultivated herbaceous plant with diverse applications in food, tea, fiber, and medicine. In this study, we present a high-quality genome assembly of H. sabdariffa using more than 33 Gb of high-fidelity (HiFi) long-read sequencing data, corresponding to ∼20× depth of the genome. We obtained 3 genome assemblies of H. sabdariffa: 1 primary and 2 partially haplotype-resolved genome assemblies. These genome assemblies exhibit N50 contig lengths of 26.25, 11.96, and 14.50 Mb, with genome coverage of 141.3, 86.0, and 88.6%, respectively. We also utilized 26 Gb of total RNA sequencing data to predict 154k, 79k, and 87k genes in the respective assemblies. The completeness of the primary genome assembly and its predicted genes was confirmed by the benchmarking universal single-copy ortholog analysis with a completeness rate of 99.3%. Based on our high-quality genomic resources, we constructed genetic networks for phenylpropanoid and flavonoid metabolism and identified candidate biosynthetic genes, which are responsible for producing key intermediates of roselle-specific medicinal natural products. Our comprehensive genomic and functional analysis opens avenues for further exploration and application of valuable natural products in H. sabdariffa.

Salmonella Typhimurium with Eight Tandem Copies of blaNDM-1 on a HI2 Plasmid.

Carbapenem-resistant Salmonella has recently aroused increasing attention. In this study, a total of four sequence type 36 Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates were consecutively isolated from an 11-month-old female patient with a gastrointestinal infection, of which one was sensitive to carbapenems and three were resistant to carbapenems. Via antibiotic susceptibility testing, a carbapenemases screening test, plasmid conjugation experiments, Illumina short-reads, and PacBio HiFi sequencing, we found that all four S. Typhimurium isolates contained a blaCTX-M-14-positive IncI1 plasmid. One carbapenem-sensitive S. Typhimurium isolate then obtained an IncHI2 plasmid carrying blaNDM-1 and an IncP plasmid without any resistance genes during the disease progression. The blaNDM-1 gene was located on a new 30 kb multiple drug resistance region, which is flanked by IS26 and TnAs2, respectively. In addition, the ST_F0903R isolate contained eight tandem copies of the ISCR1 unit (ISCR1-dsbD-trpF-ble-blaNDM-1-ISAba125Δ1), but an increase in MICs to carbapenems was not observed. Our work further provided evidence of the rapid spread and amplification of blaNDM-1 through plasmid. Prompting the recognition of carbapenem-resistant Enterobacterales and the initiation of appropriate infection control measures are essential to avoid the spread of these organisms.