A major part of any biological system on earth involves microorganisms, of which the majority are yet to be cultured. The conventional methods of culturing microbes have given fruitful outcomes yet have limitations. The curiosity for better understanding has led to the development of culture-independent molecular methods that help push aside the roadblocks of earlier methods. Metagenomics unifies the scientific community in search of a better understanding of the functioning of the ecosystem and its component organisms. This approach has opened a new paradigm in advanced research. It has brought to light the vast diversity and novelty among microbial communities and their genomes. This review focuses on the development of this field over time, the techniques and analysis of data generated through sequencing platforms, and its prominent interpretation and representation.

Skin findings can be critical to determining whether a patient with lymphangioleiomyomatosis (LAM), a progressive pulmonary disease that predominantly affects adult women, has sporadic LAM or LAM in association with tuberous sclerosis complex (TSC). Three individuals with LAM underwent evaluation for TSC-associated mucocutaneous and internal findings. We used our previously published algorithm to confirm the clinical suspicion for mosaicism and guide the selection of tissue specimens and genetic workup. Next-generation sequencing of cutaneous findings was used to confirm clinical suspicion for mosaic TSC in individuals with LAM. Two individuals previously thought to have sporadic LAM were diagnosed with mosaic TSC-associated LAM upon next-generation sequencing of unilateral angiofibromas in one and an unusual cutaneous hamartoma in the other. A third individual, diagnosed with TSC in childhood, was found to have a mosaic pathogenic variant in TSC2 in cutaneous tissue from a digit with macrodactyly. Accurate diagnosis of mosaic TSC-associated LAM may require enhanced genetic testing and is important because of the implications regarding surveillance, prognosis, and risk of transmission to offspring.

UNASSIGNED: To compare the ultrasonography and pathology features between children and adolescents with papillary thyroid carcinoma (PTC).
UNASSIGNED: A total of 53 patients who were surgically diagnosed with childhood or adolescent PTC between 2017 and 2022 were included in this study. The pre-operative ultrasonography, post-operative histology, and molecular and clinical characteristics were retrospectively analyzed.
UNASSIGNED: No differences were observed in composition, echogenicity, and shape using ultrasonography. Moreover, there was a significantly higher rate of extrathyroidal extension, punctate echogenic foci, and lymph node metastases in children compared to adolescents. The molecular analysis showed that BRAFV600E mutations are the most prevalent abnormality in adolescent PTC (12/20, 60.0%). However, they are less in childhood PTC (7/23, 30.4%). In addition, using next-generation sequencing, three cases with oncogenic fusion (one TRIM33-RET case, one CCDC6-RET case, and one STRN-ALK case) were identified in childhood PTC.
UNASSIGNED: The frequency of extrathyroidal extension, punctate echogenic foci, and lymph node metastases were higher in childhood PTC, while BRAFV600E mutations were higher in adolescent PTC.

Melanoma tumor syndromes (MTS) represent an important minority of familial melanoma cases. In these patients, the accumulation of sequence alterations in essential genes may prelude the risk of internal malignancies, in addition to melanoma. Although several host and environmental factors have been implicated in familial melanoma, the exact mechanisms of cancer predisposition-particularly in the context of mixed cancer syndromes-still remain unclear. In this paper, we review new insights into MTS and elucidate recent efforts that guide individualized prognostication and treatment for these diseases in the past quarter century.

UNASSIGNED: The study of microbial communities and their applications have been leveraged by advances in sequencing techniques and bioinformatics tools. The Oxford Nanopore Technologies long-read sequencing by nanopores provides a portable and cost-efficient platform for sequencing assays. While this opens the possibility of sequencing applications outside specialized environments and real-time analysis of data, complementing the existing efficient library preparation protocols with streamlined bioinformatic workflows is required.
UNASSIGNED: Here we present NanoRTax, a Nextflow pipeline for nanopore 16S rRNA gene amplicon data that features state-of-the-art taxonomic classification tools and real-time capability. The pipeline is paired with a web-based visual interface to enable user-friendly inspections of the experiment in progress. NanoRTax workflow and a simulated real-time analysis were used to validate the prediction of adult Intensive Care Unit patient mortality based on full-length 16S rRNA sequencing data from respiratory microbiome samples.
UNASSIGNED: This constitutes a proof-of-concept simulation study of how real-time bioinformatic workflows could be used to shorten the turnaround times in critical care settings and provides an instrument for future research on early-response strategies for sepsis.

Increasing globalization, agricultural intensification, urbanization, and climatic changes have resulted in a significant recent increase in emerging infectious zoonotic diseases. Zoonotic diseases are becoming more common, so innovative, effective, and integrative research is required to better understand their transmission, ecological implications, and dynamics at wildlife-human interfaces. High-throughput sequencing (HTS) methodologies have enormous potential for unraveling these contingencies and improving our understanding, but they are only now beginning to be realized in livestock research. This study investigates the current state of use of sequencing technologies in the detection of livestock pathogens such as bovine, dogs (Canis lupus familiaris), sheep (Ovis aries), pigs (Sus scrofa), horses (Equus caballus), chicken (Gallus gallus domesticus), and ducks (Anatidae) as well as how it can improve the monitoring and detection of zoonotic infections. We also described several high-throughput sequencing approaches for improved detection of known, unknown, and emerging infectious agents, resulting in better infectious disease diagnosis, as well as surveillance of zoonotic infectious diseases. In the coming years, the continued advancement of sequencing technologies will improve livestock research and hasten the development of various new genomic and technological studies on farm animals.

Many toxins are life-threatening to both animals and humans. However, specific antidotes are not available for most of those toxins. The molecular mechanisms underlying the toxicology of well-known toxins are not yet fully characterized. Recently, the advance in CRISPR-Cas9 technologies has greatly accelerated the process of revealing the toxic mechanisms of some common toxins on hosts from a genome-wide perspective. The high-throughput CRISPR screen has made it feasible to untangle complicated interactions between a particular toxin and its corresponding targeting tissue(s). In this review, we present an overview of recent advances in molecular dissection of toxins\' cytotoxicity by using genome-wide CRISPR screens, summarize the components essential for toxin-specific CRISPR screens, and propose new strategies for future research.

UNASSIGNED: Autosomal recessive Gaucher disease (GD) is likely underdiagnosed in many countries. Because the number of diagnosed GD patients in Finland is relatively low, and the true prevalence is currently not known, it was hypothesized that undiagnosed GD patients may exist in Finland. Our previous study demonstrated the applicability of Gaucher Earlier Diagnosis Consensus point-scoring system (GED-C PSS; Mehta et al., 2019) and Finnish biobank data and specimens in the automated point scoring of large populations. An indicative point-score range for Finnish GD patients was determined, but undiagnosed patients were not identified partly due to high number of high-score subjects in combination with a lack of suitable samples for diagnostics in the assessed biobank population. The current study extended the screening to another biobank and evaluated the feasibility of utilising the automated GED-C PSS in conjunction with small nucleotide polymorphism (SNP) chip genotype data from the FinnGen study of biobank sample donors in the identification of undiagnosed GD patients in Finland. Furthermore, the applicability of FFPE tissues and DNA restoration in the next-generation sequencing (NGS) of the GBA gene were tested.
UNASSIGNED: Previously diagnosed Finnish GD patients eligible to the study, and up to 45,100 sample donors in Helsinki Biobank (HBB) were point scored. The GED-C point scoring, adjusted to local data, was automated, but also partly manually verified for GD patients. The SNP chip genotype data for rare GBA variants was visually assessed. FFPE tissues of GD patients were obtained from HBB and Biobank Borealis of Northern Finland (BB).
UNASSIGNED: Three previously diagnosed GD patients and one patient previously treated for GD-related features were included. A genetic diagnosis was confirmed for the patient treated for GD-related features. The GED-C point score of the GD patients was 12.5-22.5 in the current study. The score in eight Finnish GD patients of the previous and the current study is thus 6-22.5 points per patient. In the automated point scoring of the HBB subpopulation (N ≈ 45,100), the overall scores ranged from 0 to 17.5, with 0.77% (346/45,100) of the subjects having ≥10 points. The analysis of SNP chip genotype data was able to identify the diagnosed GD patients, but potential undiagnosed patients with the GED-C score and/or the GBA genotype indicative of GD were not discovered. Restoration of the FFPE tissue DNA improved the quality of the GBA NGS, and pathogenic GBA variants were confirmed in five out of six unrestored and in all four restored FFPE DNA samples.
UNASSIGNED: These findings imply that the prevalence of diagnosed patients (~1:325,000) may indeed correspond the true prevalence of GD in Finland. The SNP chip genotype data is a valuable tool that complements the screening with the GED-C PSS, especially if the genotyping pipeline is tuned for rare variants. These proof-of-concept biobank tools can be adapted to other rare genetic diseases.

The viral genome quasispecies composition of hepatitis C virus (HCV) could have important implications to viral pathogenesis and resistance to anti-viral treatment. The purpose of the present study was to profile the HCV RNA quasispecies. We developed a strategy to determine the full-length HCV genome sequences co-existing within a single patient serum by using next-generation sequencing technologies. The isolated viral clones were divided into the groups that can be distinguished by core amino acid 70 substitution. Subsequently, we determined HCV full-length genome sequences of three independent dominant species co-existing in the sequential serum with a 7-year interval. From phylogenetic analysis, these dominant species evolved independently. Our study demonstrated that multiple dominant species co-existed in patient sera and evolved independently.

RNA sequence data are commonly summarized as read counts. By contrast, so far there is no alternative to genotype calling for investigating the relationship between genetic variants determined by next-generation sequencing (NGS) and a phenotype of interest. Here we propose and evaluate the direct analysis of allele counts for genetic association tests. Specifically, we assess the potential advantage of the ratio of alternative allele counts to the total number of reads aligned at a specific position of the genome (coverage) over called genotypes. We simulated association studies based on NGS data from HapMap individuals. Genotype quality scores and allele counts were simulated using NGS data from the Personal Genome Project. Real data from the 1000 Genomes Project was also used to compare the two competing approaches. The average proportions of probability values lower or equal to 0.05 amounted to 0.0496 for called genotypes and 0.0485 for the ratio of alternative allele counts to coverage in the null scenario, and to 0.69 for called genotypes and 0.75 for the ratio of alternative allele counts to coverage in the alternative scenario (9% power increase). The advantage in statistical power of the novel approach increased with decreasing coverage, with decreasing genotype quality and with decreasing allele frequency - 124% power increase for variants with a minor allele frequency lower than 0.05. We provide computer code in R to implement the novel approach, which does not preclude the use of complementary data quality filters before or after identification of the most promising association signals.
UNASSIGNED: Genetic association tests usually rely on called genotypes. We postulate here that the direct analysis of allele counts from sequence data improves the quality of statistical inference. To evaluate this hypothesis, we investigate simulated and real data using distinct statistical approaches. We demonstrate that association tests based on allele counts rather than called genotypes achieve higher statistical power with controlled type I error rates.