Noninvasive Prenatal Screening for Common Fetal Aneuploidies Using Single-Molecule Sequencing.

Amplification biases caused by next-generation sequencing (NGS) for noninvasive prenatal screening (NIPS) may be reduced using single-molecule sequencing (SMS), during which PCR is omitted. Therefore, the performance of SMS-based NIPS was evaluated. We used SMS-based NIPS to screen for common fetal aneuploidies in 477 pregnant women. The sensitivity, specificity, positive predictive value, and negative predictive value were estimated. The GC-induced bias was compared between the SMS- and NGS-based NIPS methods. Notably, a sensitivity of 100% was achieved for fetal trisomy 13 (T13), trisomy 18 (T18), and trisomy 21 (T21). The positive predictive value was 46.15% for T13, 96.77% for T18, and 99.07% for T21. The overall specificity was 100% (334/334). Compared with NGS, SMS (without PCR) had less GC bias, a better distinction between T21 or T18 and euploidies, and better diagnostic performance. Overall, our results suggest that SMS improves the performance of NIPS for common fetal aneuploidies by reducing the GC bias introduced during library preparation and sequencing.

Comparative performance of the GenoLab M and NovaSeq 6000 sequencing platforms for transcriptome and LncRNA analysis.

BACKGROUND: GenoLab M is a recently established next-generation sequencing platform from GeneMind Biosciences. Presently, Illumina sequencers are the globally leading sequencing platform in the next-generation sequencing market. Here, we present the first report to compare the transcriptome and LncRNA sequencing data of the GenoLab M sequencer to NovaSeq 6000 platform in various types of analysis. RESULTS: We tested 16 libraries in three species using various library kits from different companies. We compared the data quality, genes expression, alternatively spliced (AS) events, single nucleotide polymorphism (SNP), and insertions-deletions (InDel) between two sequencing platforms. The data suggested that platforms have comparable sensitivity and accuracy in terms of quantification of gene expression levels with technical compatibility. CONCLUSIONS: Genolab M is a promising next-generation sequencing platform for transcriptomics and LncRNA studies with high performance at low costs.

A Two-color Single-molecule Sequencing Platform and Its Clinical Applications.

DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years. In this study, we developed a single-molecule desktop sequencer, GenoCare 1600 (GenoCare), which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry, making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use. Using the GenoCare platform, we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%. We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019 (COVID-19) samples from throat swabs. Our findings indicate that the GenoCare platform allows for microbial quantitation, sensitive identification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and accurate detection of virus mutations, as confirmed by Sanger sequencing, demonstrating its remarkable potential in clinical application.

Evaluation of environmental factors and microbial community structure in an important drinking-water reservoir across seasons.

The composition of microbial communities varies in water and sediments, and changes in environmental factors have major effects on microbiomes. Here, we characterized variations in microbial communities and physicochemical factors at two sites in a large subtropical drinking water reservoir in southern China. The microbiomes of all sites, including the diversity and abundance of microbial species, were determined via metagenomics, and the relationships between microbiomes and physicochemical factors were determined via redundancy analysis. The dominant species in sediment and water samples differed; Dinobryon sp. LO226KS and Dinobryon divergens were dominant in sediment samples, whereas Candidatus Fonsibacter ubiquis and Microcystis elabens were dominant in water. The diversity was also significantly different in microbial alpha diversity between water and sediment habitats (p < 0.01). The trophic level index (TLI) was the major factor affecting the microbial community in water samples; Mycolicibacterium litorale and Mycolicibacterium phlei were significantly positively related to TLI. Furthermore, we also studied the distribution of algal toxin-encoding genes and antibiotic-resistant genes (ARGs) in the reservoir. It found that water samples contained more phycotoxin genes, with the cylindrospermopsin gene cluster most abundant. We found three genera highly related to cylindrospermopsin and explored a new cyanobacteria Aphanocapsa montana that may produce cylindrospermopsin based on the correlation through network analysis. The multidrug resistance gene was the most abundant ARG, while the relationship between ARGs and bacteria in sediment samples was more complicated than in water. The results of this study enhance our understanding of the effects of environmental factors on microbiomes. In conclusion, research on the properties, including profiles of algal toxin-encoding genes and ARGs, and microbial communities can aid water quality monitoring and conservation.

Characterizing the gene mutations associated with resistance to gatifloxacin in Mycobacterium tuberculosis through whole-genome sequencing.

OBJECTIVES: Gatifloxacin (GAT), a fourth-generation fluoroquinolone (FQ), is used to treat drug-resistant tuberculosis. Although DNA gyrase mutations are the leading cause of FQ resistance, mutations conferring resistance to GAT remain inadequately characterized. METHODS: GAT-resistant mutants were selected from 7H10 agar plates containing 0.5 mg/L GAT (critical concentration). Mutations involved in GAT resistance were identified through whole-genome sequencing. RESULTS: In total, 123 isolates demonstrated resistance to GAT. Among these isolates, 55.3% (68/123) had gyrA gene mutations [G280A (D94N), A281G (D94G), G280T (D94Y) and G262T (G88C)]. The remainder (44.7%, 55/123) harboured gyrB gene mutations [A1495G (N499D), C1497A (N499K), C1497G (N499K) and A1503C (E501D)]. CONCLUSIONS: Mutations in the gyrA and gyrB genes are the main mechanisms of GAT resistance. These findings provide new insight into GAT resistance, and contribute to molecular diagnosis of GAT resistance in the clinical setting.

Fraternal twins with Phelan-McDermid syndrome not involving the SHANK3 gene: case report and literature review.

BACKGROUND: Phelan-McDermid syndrome (PMS, OMIM#606232), or 22q13 deletion syndrome, is a rare genetic disorder caused by deletion of the distal long arm of chromosome 22 with a variety of clinical features that display considerably heterogeneous degrees of severity. The SHANK3 gene is understood to be the critical gene for the neurological features of this syndrome. CASE PRESENTATION: We describe one pair of boy-girl twins with a 22q13 deletion not involving the SHANK3 gene. Interestingly, the clinical and molecular findings of the two patients were identical, likely resulting from germline mosaicism in a parent. The boy-girl twins showed intellectual disability, speech absence, facial dysmorphism, cyanosis, large fleshy hands and feet, dysplastic fingernails and abnormal behaviors, and third-generation sequencing showed an identical de novo interstitial deletion of 6.0 Mb in the 22q13.31-q13.33 region. CONCLUSIONS: Our case suggests that prenatal diagnosis is essential for normal parents with affected children due to the theoretical possibility of parental germline mosaicism. Our results also indicated that other genes located in the 22q13 region may have a role in explaining symptoms in individuals with PMS. In particular, we propose that four candidate genes, CELSR1, ATXN10, FBLN1 and WNT7B, may also be involved in the etiology of the clinical features of PMS. However, more studies of smaller interstitial deletions with 22q13 are needed to corroborate our hypothesis and better define the genotype-phenotype correlation. Our findings contribute to a more comprehensive understanding of PMS.

Statistical analysis of mutant allele frequency level of circulating cell-free DNA and blood cells in healthy individuals.

Cell-free DNA (cfDNA) in plasma has emerged as a potential important biomarker in clinical diagnostics, particularly in cancer. However, somatic mutations are also commonly found in healthy individuals, which interfere with the effectiveness for cancer diagnostics. This study examined the background somatic mutations in white blood cells (WBC) and cfDNA in healthy controls based on sequencing data from 821 non-cancer individuals and several cancer samples with the aim of understanding the patterns of mutations detected in cfDNA. We determined the mutation allele frequencies in both WBC and cfDNA using a panel of 50 cancer-associated genes that covers 20 K-nucleotide region and ultra-deep sequencing with average depth >40000-fold. Our results showed that most of the mutations in cfDNA were highly correlated to WBC. We also observed that the NPM1 gene was the most frequently mutated gene in both WBC and cfDNA. Our study highlighted the importance of sequencing both cfDNA and WBC to improve the sensitivity and accuracy for calling cancer-related mutations from circulating tumour DNA, and shedded light on developing a strategy for early cancer diagnosis by cfDNA sequencing.

Single molecule sequencing of the M13 virus genome without amplification.

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.