BACKGROUND AND OBJECTIVE

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of COVID-19 has significantly challenged the public health landscape in late 2019. After almost 3 years of the first ever SARS-CoV-2 case, the World Health Organization (WHO) declared the end of this global health emergency in May 2023. Although, despite the subsequent drop of COVID-19 cases, the SARS-CoV-2 infection still exhibited multiple waves of infection, primarily attributed to the appearance of new variants. Five of these variants have been classified as Variants of Concern (VOC): Alpha, Beta, Gamma, Delta, and the most recent, Omicron. Therefore, the development of methods for the timely and accurate detection of viral variants remains fundamental, ensuring an ongoing and effective response to the disease. This study aims to evaluate the feasibility of the application of an in-house approach in genomic surveillance for the detection of SARS-CoV-2 variants using in silico designed primers.

METHODS

The primers used for the study were particularly designed based on conserved regions of certain genes in the virus, targeting distinct mutations found in known variants of SARS-CoV-2. Viral RNA extracts from nasopharyngeal samples (n=14) were subjected to quantitative and qualitative tests (Nanodrop and AGE). Selected samples were then analyzed by RT-PCR and amplicons were submitted for sequencing. Sequence alignment analysis was carried out to identify the prevailing COVID-19 variant present in the sample population.

RESULTS

The study findings demonstrated that the in-house method was able to successfully amplify conserved sequences (spike, envelope, membrane, ORF1ab) and enabled identification of the circulating SARS-CoV-2 variant among the samples. Majority of the samples were identified as Omicron variant. Three out of four designed primers effectively bound into the conserved sequence of target genes present in the sample, revealing the specific SARSCoV-2 variant. The detected mutations characterized for Omicron found in the identified lineages included K417N, S477N, and P681H which were also identified as mutations of interest. Furthermore, identification of the B.1.448 lineage which was not classified in any known variant also provided the potential of the developed in-house method in detecting unknown variants of COVID-19.

CONCLUSION

Among the five VOCs, Omicron is the most prevalent and dominant variant. The in-house direct PCR product sequencing surveillance (DPPSS) method provided an alternative platform for SAR-CoV-2 variant analysis which is accessible and affordable than the conventional diagnostic surveillance methods and the whole genome sequencing. Further evaluation and improvements on the oligonucleotide primers may offer significant contribution to the development of a specific and direct PCRbased detection of new emerging COVID-19 variants.

Sars-cov-2 ; Polymerase Chain Reaction ; Dna Primers ; Oligonucleotide Primers ; Computer Simulation ; Conserved Sequence ; Coronavirus ; Covid-19 ; Disease ; Emergencies ; Evaluation Studies As Topic ; Genes ; Genome ; Global Health ; Health ; Identification (psychology) ; Infection ; Infections ; Membranes ; Methods ; Mutation ; Oligonucleotides ; Organizations ; Population ; Public Health ; Rna ; Rna, Viral ; Sars Virus ; Sequence Alignment ; Severe Acute Respiratory Syndrome ; Syndrome ; Viruses ; Whole Genome Sequencing ; World Health Organization

OBJECTIVE: To re-analyze a likely pathogenic variant in the Xq28 region identified by copy number variation sequencing (CNV-seq) through whole genome sequencing (WGS). METHODS: A fetus found to harbor a duplication in the Xq28 region by CNV-seq at Shengjing Hospital Affiliated to China Medical University in May 2023 was selected as the study subject. WGS was carried out for the fetus and its parents. Bioinformatic software was used to analyze the chromosomal structure and CNVs. Quantitative PCR (qPCR) was applied to determine the expression level of the MECP2 gene. This study has been approved by the Ethics Committee of Shengjing Hospital (Ethic No. 2013PS33K). RESULTS: A duplication (ChrX:153302641_153503563) and four breakpoints were identified on the X chromosome of the fetus' father. Bioinformatic analysis revealed that the duplicated region has involved exons 1 to 3 and part of the 5'-UTR of the MECP2 gene, which was inserted into the Xp11 region. Additionally, an inversion was detected in the Xp11 region adjacent to the duplicated segment. RT-PCR results showed normal level of MECP2 mRNA expression. The Xq28 duplication has not encompassed the entire MECP2 gene, nor disrupted its structure or altered its expression. CONCLUSION WGS has enabled more precise diagnosis of chromosomal structural variants and provided guidance for accurate genetic counseling for the affected families.
Humans ; Female ; Chromosomes, Human, X/genetics* ; DNA Copy Number Variations/genetics* ; Whole Genome Sequencing/methods* ; Methyl-CpG-Binding Protein 2/genetics* ; Pregnancy ; Male ; Adult

OBJECTIVE: To explore the genetic etiology of a Chinese pedigree with recurrent Joubert syndrome with negative results by whole-exome sequencing in the prior proband. METHODS: Chinese pedigree which opted elective abortion at the Women and Children's Hospital Affiliated to Chongqing Medical University in December 2024 was selected as the study subject. Whole-genome sequencing was carried out on fetal tissue after termination of pregnancy. Candidate variants were validated by Sanger sequencing and interpreted, while non-coding variant was analyzed using in silico prediction tools. RNA sequencing and cDNA sequencing were conducted on fetal brain tissue. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.2024YL045-02). RESULTS: Both the fetus and the affected child were found to harbor compound heterozygous variants of the CEP290 gene, namely c.7341dup (p.Leu2448fs*8) (pathogenic, maternally inherited) and c.1523-408G>A (likely pathogenic, paternally inherited). Both in silico analysis and fetal brain RNA sequencing confirmed aberrant RNA splicing caused by the intronic variant. CONCLUSION This case has highlighted the value of combining whole-genome sequencing with RNA functional validation. Above results not only enriched the spectrum of CEP290 gene mutations but also underscored its diagnostic value in resolving complex prenatal cases, providing critical clues for the prenatal diagnosis and recurrence risk assessment in genetic counseling.
Female ; Humans ; Pregnancy ; Abnormalities, Multiple/genetics* ; Antigens, Neoplasm/genetics* ; Cell Cycle Proteins/genetics* ; Cerebellum/abnormalities* ; Cytoskeletal Proteins/genetics* ; Eye Abnormalities/genetics* ; Introns/genetics* ; Kidney Diseases, Cystic/diagnosis* ; Pedigree ; Retina/abnormalities* ; Sequence Analysis, RNA/methods* ; Whole Genome Sequencing/methods* ; Child

OBJECTIVE: To explore the genetic characteristics and pathogenesis for a child with mosaicism trisomy 21 and Congenital leukemia (CL). METHODS: A child who was admitted to Ningbo Women and Children's Hospital in March 2023 was selected as the study subject. A retrospective analysis was carried out on the clinical data, laboratory test results, immunophenotyping, and genetic characteristics of the child. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: EC2024-063). RESULTS: Whole genome sequencing (WGS) revealed that the child has mosaicism trisomy of chromosome 21, with a ratio of approximately 74%. In addition, copy number variations involving multiple OMIM genes that could explain his clinical phenotype were detected and rated as pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). No pathogenic variant was detected with the GATA1 gene. Blood immune typing of the child conformed to the immunophenotype of acute myeloid leukemia. CONCLUSION For children with trisomy 21, even in the absence of GATA1 gene variants, the occurrence of CL should be monitored, and early diagnosis and treatment are of great significance for improving the prognosis.
Child, Preschool ; Humans ; DNA Copy Number Variations/genetics* ; Down Syndrome/genetics* ; GATA1 Transcription Factor/genetics* ; Leukemia/congenital* ; Mosaicism ; Mutation ; Retrospective Studies ; Whole Genome Sequencing

OBJECTIVES: To explore the molecular characteristics of Staphylococcus aureus (S. aureus) in children, and to compare the molecular characteristics of different types of strains (infection and colonization strains) so as to reveal pathogenic molecular markers of S. aureus. METHODS: A cross-sectional study design was used to conduct nasopharyngeal swab sampling from healthy children in the community and clinical samples from infected children in the hospital. Whole genome sequencing was used to detect antibiotic resistance genes and virulence genes. A random forest method to used to screen pathogenic markers. RESULTS: A total of 512 S. aureus strains were detected, including 272 infection strains and 240 colonization strains. For virulence genes, the carrying rates of enterotoxin genes (seb and sep), extracellular enzyme coding genes (splA, splB, splE and edinC), leukocytotoxin genes (lukD, lukE, lukF-PV and lukS-PV) and epidermal exfoliating genes (eta and etb) in infection strains were higher than those in colonization strains. But the carrying rates of enterotoxin genes (sec, sec3, seg, seh, sei, sel, sem, sen, seo and seu) were lower in infection strains than in colonization strains (P<0.05). For antibiotic resistance genes, the carrying rates of lnuA, lnuG, aadD, tetK and dfrG were significantly higher in infection strains than in colonization strains (P<0.05). The accuracy of cross-validation of the random forest model for screening pathogenic markers of S. aureus before and after screening was 69% and 68%, respectively, and the area under the curve was 0.75 and 0.70, respectively. The random forest model finally screened out 16 pathogenic markers (sem, etb, splE, sep, ser, mecA, lnuA, sea, blaZ, cat(pC233), blaTEm-1A, aph(3')-III, ermB, ermA, ant(9)-Ia and ant(6)-Ia). The top five variables in the variable importance ranking were sem (OR=0.40), etb (OR=3.95), splE (OR=1.68), sep (OR=3.97), and ser (OR=1.68). CONCLUSIONS The random forest model can screen out pathogenic markers of S. aureus and exhibits a superior predictive performance, providing genetic evidence for tracing highly pathogenic S. aureus and conducting precise targeted interventions.
Child ; Humans ; Staphylococcus aureus/genetics* ; Cross-Sectional Studies ; Enterotoxins/genetics* ; Staphylococcal Infections ; Whole Genome Sequencing

Incarvillea younghusbandii Sprague is a traditional tonic herb. The roots are used as herbal medicine for nourishing and strengthening, as well as treating postpartum milk deficiency and weakness. In this study, the chloroplast genome of I. younghusbandii was sequenced and assembled by the high-throughput sequencing technology. The sequence characteristics, sequence repeats, codon usage bias, phylogenetic relationships and estimated divergence time of I. younghusbandii were analyzed. The 159 323 bp sequence contained a large single copy (80 197 bp), a small single copy (9 030 bp) and two inverted repeat sequences (35 048 bp). It contained 120 genes, including 77 protein coding genes, 8 ribosomal RNA genes and 35 transfer RNA genes. AAA was the most frequent codon in the chloroplast coding sequence of I. younghusbandii. A total of 42 simple sequence repeats were identified in the chloroplast genome. Phylogenetic analysis revealed I. younghusbandii was mostly like its taxonomically close relative Incarvillea compacta. The divergence between I. younghusbandii and I. compacta was dated to 4.66 million years ago. This study was significant for the scientific conservation and development of resources related to I. compacta. It also provides a basic genetic resource for the subsequent species identification of the genus Incarvillea, and the population genetic diversity study of Bignoniaceae.
Phylogeny ; Molecular Sequence Annotation ; Genome, Chloroplast ; Sequence Analysis, DNA ; Whole Genome Sequencing

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.
Phylogeny ; Atractylodes/genetics* ; Genome, Chloroplast ; Whole Genome Sequencing ; Microsatellite Repeats ; Lamiales

OBJECTIVE: To explore the association between de novo mutations (DNM) and non-syndromic cleft lip with or without palate (NSCL/P) using case-parent trio design. METHODS: Whole-exome sequencing was conducted for twenty-two NSCL/P trios and Genome Analysis ToolKit (GATK) was used to identify DNM by comparing the alleles of the cases and their parents. Information of predictable functions was annotated to the locus with SnpEff. Enrichment analysis for DNM was conducted to test the difference between the actual number and the expected number of DNM, and to explore whether there were genes with more DNM than expected. NSCL/P-related genes indicated by previous studies with solid evidence were selected by literature reviewing. Protein-protein interactions analysis was conducted among the genes with protein-altering DNM and NSCL/P-related genes. R package "denovolyzeR" was used for the enrichment analysis (Bonferroni correction: P=0.05/n, n is the number of genes in the whole genome range). Protein-protein interactions among genes with DNM and genes with solid evidence on the risk factors of NSCL/P were predicted depending on the information provided by STRING database. RESULTS: A total of 339 908 SNPs were qualified for the subsequent analysis after quality control. The number of high confident DNM identified by GATK was 345. Among those DNM, forty-four DNM were missense mutations, one DNM was nonsense mutation, two DNM were splicing site mutations, twenty DNM were synonymous mutations and others were located in intron or intergenic regions. The results of enrichment analysis showed that the number of protein-altering DNM on the exome regions was larger than expected (P < 0.05), and five genes (KRTCAP2, HMCN2, ANKRD36C, ADGRL2 and DIPK2A) had more DNM than expected (P < 0.05/(2×19 618)). Protein-protein interaction analysis was conducted among forty-six genes with protein-altering DNM and thirteen genes associated with NSCL/P selected by literature reviewing. Six pairs of interactions occurred between the genes with DNM and known NSCL/P-related genes. The score measuring the confidence level of the predicted interaction between RGPD4 and SUMO1 was 0.868, which was higher than the scores for other pairs of genes. CONCLUSION Our study provided novel insights into the development of NSCL/P and demonstrated that functional analyses of genes carrying DNM were warranted to understand the genetic architecture of complex diseases.
Asians ; Case-Control Studies ; Cleft Lip/genetics* ; Cleft Palate/genetics* ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Genotype ; Humans ; Mutation ; Parents ; Polymorphism, Single Nucleotide ; Whole Exome Sequencing

COVID-19/genetics* ; Genome, Viral/genetics* ; Humans ; RNA, Viral/genetics* ; RNA-Seq ; SARS-CoV-2/genetics* ; Whole Genome Sequencing

Abnormalities, Multiple/genetics* ; Costello Syndrome ; Humans ; Whole Genome Sequencing