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PubMed2024

Application of third-generation sequencing technology for identifying rare α- and β-globin gene variants in a Southeast Chinese region.

BACKGROUND: Third-generation sequencing (TGS) based on long-read technology has been gradually used in identifying thalassemia and hemoglobin (Hb) variants. The aim of the present study was to explore genotype varieties of thalassemia and Hb variants in Quanzhou region of Southeast China by TGS. METHODS: Included in this study were 6,174 subjects with thalassemia traits from Quanzhou region of Southeast China. All of them underwent common thalassemia gene testing using the DNA reverse dot-blot hybridization technology. Subjects who were suspected as rare thalassemia carriers were further subjected to TGS to identify rare or novel α- and β-globin gene variants, and the results were verified by Sanger sequencing and/or gap PCR. RESULTS: Of the 6,174 included subjects, 2,390 (38.71%) were identified as α- and β-globin gene mutation carriers, including 40 carrying rare or novel α- and β-thalassemia mutations. The αCD30(-GAG)α and Hb Lepore-Boston-Washington were first reported in Fujian province Southeast China. Moreover, the βCD15(TGG> TAG), βIVS-II-761, β0-Filipino(~ 45 kb deletion), and Hb Lepore-Quanzhou were first identified in the Chinese population. In addition, 35 cases of Hb variants were detected, the rare Hb variants of Hb Jilin and Hb Beijing were first reported in Fujian province of China. Among them, one case with compound αααanti3.7 and Hb G-Honolulu variants was identified in this study. CONCLUSION: Our findings may provide valuable data for enriching the spectrum of thalassemia and highlight the clinical application value of TGS-based α- and β-globin genetic testing.

Humans↗
PubMed2025

Whole genome study and construction of SHERLOCK detection method for endemic strains of Burkholderia pseudomallei in Hainan based on third-generation sequencing.

UNLABELLED: Burkholderia pseudomallei (Bp) is a gram-negative bacterium found in soil and surface water. It is also the pathogen that causes melioidosis disease in humans and animals. This study aimed to obtain the whole genome sequence of the endemic strain of Bp in Hainan, using third-generation sequencing (TGS) technology, and elucidate the genome structure, function, and genetic evolution. Additionally, the study aimed to achieve rapid and specific identification of these endemic strains using specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) detection technology, providing a new strategy for the early diagnosis of melioidosis. Utilizing the PacBio platform for TGS technology, we completed whole genome sequencing of 16 Bp strains from Hainan. High-precision and complete genome sequences were obtained through quality control and genome assembly of the sequencing data. Additionally, we established a nucleic acid detection technology platform based on SHERLOCK, which could be completed from nucleic acid extraction to result reading within 1-2 hours, demonstrating good sensitivity and specificity (both are 100%). The lateral chromatography strip method does not require special equipment and holds promise as an immediate screening method for the early diagnosis of melioidosis. IMPORTANCE: Melioidosis is a highly pathogenic infectious disease caused by a gram-negative bacterium of Burkholderia pseudomallei (Bp). The traditional gold standard for diagnosing melioidosis is still isolation and culture from clinical samples. Although this method has high specificity, it has low sensitivity and is time-consuming, which often leads to misdiagnosis or missed diagnosis of melioidosis, affecting subsequent treatment. In this study, recombinase polymerase amplification technology and clustered regularly interspaced short palindromic repeats/Cas13a technology were combined to establish the Specific High-sensitivity Enzymatic Reporter Unlocking detection technology, which can achieve rapid and accurate identification of Bp, providing a new method for the early diagnosis of melioidosis.

Burkholderia pseudomallei↗
PubMed2025

SMART-RNA-Metavirome: a practical RNA metavirome platform compatible with high-throughput sequencing of both short and long reads.

BACKGROUND: The RNA virosphere's extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens, including co-infections. However, enriching low-abundance viral sequences in RNA metaviromics, particularly in the preparation of cDNA libraries and their compatibility with next-generation sequencing (NGS) and third-generation sequencing (TGS), remains challenging. Therefore, our objective is to develop and systematically assess a practical RNA metavirome methodology specifically tailored for the enrichment of low-abundance viral sequences within samples. METHODS: We developed the SMART-RNA-Metavirome platform, integrating SMART-9n library preparation with NGS and TGS technologies. Total RNA was extracted from two field-collected wild Aedes albopictus pools, along with one laboratory-infected Ae. albopictus pool harboring dengue virus (DENV). This RNA was subjected to reverse transcription using both this optimized protocol and random primer-based methods, followed by high-throughput sequencing on Illumina, Oxford Nanopore, and QitanTech Nanopore technologies. Welch's t-test was employed for comparative analysis of the subsequent RNA metavirome data, specifically to evaluate differences in viral species composition and abundance of viral reads between experimental groups. Furthermore, the effectiveness of this platform was systematically validated via RT-qPCR and SMART-RNA-Metavirome-based Oxford Nanopore sequencing across multiple sample types, including mosquito specimens from DENV-infected Ae. albopictus, serum samples from dengue patients and viral isolates of Japanese encephalitis virus (JEV) and Zika virus (ZIKV). RESULTS: The SMART-RNA-Metavirome platform has been systematically validated to excel in enriching the composition and diversity of the RNA virome (P = 0.04), providing sufficient coverage for the complete reconstruction of viral genomes. When employed in the detection of DENV-infected Ae. albopictus, clinical serum samples, and viral isolates of JEV and ZIKV, this technique exhibits a robust correlation with RT-qPCR (r2 > 0.95). Notably, it demonstrates exceptional sensitivity, ensuring sufficient coverage even in samples of DENV-infected Ae. albopictus with a Ct-value of 35.3, attaining an impressive 99.88% genome coverage. Furthermore, this platform possesses the capability to identify virus species and determine their serotypes. CONCLUSIONS: In our study, the SMART-RNA-Metavirome platform outperforms traditional methods, enriching RNA virome composition and diversity, enabling practical compatibility with both NGS and TGS technologies. It demonstrates significant proficiency in detecting both known and unknown arboviruses, even in low-titer samples such as those from wild mosquitoes and clinical sera. This platform facilitates comprehensive monitoring, risk assessment, and early warning of RNA virus transmissions, enhancing our understanding of RNA virome diversity and ecological patterns.

High-Throughput Nucleotide Sequencing↗
PubMed2025

Allele Level Sequencing of Killer Cell Immunoglobulin-Like Receptor Genes Using Oxford Nanopore Long Read Sequencing.

The human Killer cell Immunoglobulin-like Receptor (KIR) genes, found on chromosome 19, encode for cell surface protein receptors that, through interaction with their ligand, modulate the action of Natural Killer (NK) cells and some subsets of T lymphocytes. KIR genes exhibit extensive variation through variable gene content, copy number, and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant, and infectious disease progression. KIR gene content has been used in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. With the introduction of massively parallel clonal next generation sequencing and single molecule long read third generation sequencing, allele level determination of KIR genotypes has become feasible. We describe a method for amplicon-based long read sequencing on the Oxford Nanopore Technologies platform that provides largely unambiguous allele level typing of KIR genes. The method was validated using DNA extracted from 48 10th International Histocompatibility Workshop (IHWS) cell lines with previously published allele level KIR genotypes and 176 Western Australian samples previously tested for the presence or absence of KIR genes. Our long-read sequencing method was able to accurately determine KIR alleles with an overall concordance of 97%-99% with the published data. Importantly, phasing ambiguity caused by the inability to phase heterozygous base positions over long stretches of gene sequence was resolved in several samples. Thus, our long read PCR sequencing strategy can be used to determine KIR genotypes at allele resolution level.

Humans↗