Sequencing Protocol Research Articles

Personalized Residual Disease Detection in Acute Myeloid Leukemia with a Multiplexed Genomic Fingerprint Predicts Patient Outcomes

Background: Acute myeloid leukemia (AML) is the most common leukemia in adults. While therapeutic options for AML have continued to improve, nearly half of all patients who achieve clinical remission will relapse within several years after diagnosis. Risk factors for relapse include age, cytogenetics, specific gene mutations, and the detection of residual disease after treatment. Current molecular approaches for measuring residual disease are designed primarily around a single gene. This limits both the sensitivity and the applicability of the assay to all patients, as AML is a highly heterogenous genetic disease. In this study, we demonstrate the utility of a comprehensive, personalized, multiplexed genomic assay that exhibits high sensitivity, excellent quantitation, and promising performance across tissue types. This approach enables the serial, non-invasive monitoring of patients with AML and the prediction of patient outcome. Methods: Serial samples of bone marrow, peripheral blood, and urine were collected at multiple time points from 12 patients with AML who were treated with standard-of-care therapy. Time points range from presentation to relapse, with a median of 4 time points per patient (range of 2-10). A median of 7 samples were collected per patient (range of 2 to 16). DNA was extracted from the peripheral blood mononuclear cells (PBMCs), the cell-free fraction of peripheral blood, bone marrow mononuclear cells, and the cell-free fraction of urine. Deep whole genome sequencing of paired presentation and remission samples identified hundreds of candidate leukemia-associated somatic single-nucleotide variants comprising the personalized genomic fingerprint of each patient's disease. A representative set of variants (n=30-50) was selected for each patient, and a personalized set of primers was designed for the Multiplex Accurate Sensitive Quantification (MASQ) next-generation sequencing protocol. The sensitivity of this assay allows for single variants to be quantified down to at least 1 in 100,000, resulting in an overall sensitivity below 1 in 1 million for residual disease detection. Quantification of the individual variants from the fingerprint was evaluated in samples of all tissue types collected (bone marrow, blood cells, blood cell-free DNA, and urine cell-free DNA) and all time points collected (presentation, post-induction, post-consolidation, and follow-up visits). Results: Serial monitoring of the leukemia genomic fingerprint enables risk assessment after each stage of treatment. Using Multiplex Accurate Sensitive Quantification (MASQ), we detected leukemia-associated variants in the blood and bone marrow of all patients during remission, at levels ranging from less than 10^-6 to 10^-2. We find consistent low signal, less than 10^-4, in both bone marrow and blood in patients with good prognosis. In contrast, we find high signal in remission, greater than 10^-3, in patients with poor prognosis. In the analysis of serial samples, frequencies drop over time with additional treatment and increase preceding clinical identification of relapse. Figure 1A shows the trajectory of 50 variants over 5 time points in PBMCs, preceding relapse. The overall signal increases at 7 months prior to clinical relapse and again at 5 months prior to clinical relapse. Figure 1B shows a comparison of the aggregate estimates of the leukemia genomic fingerprint in the cellular and cell-free components of the blood. While total DNA yields from the cell-free fraction of blood are lower than from PBMCs, we find that the frequency of the leukemia genomic fingerprint in the two sources of DNA is highly similar. Bone marrow and peripheral blood also show highly similar measurements of residual disease, suggesting that non-invasive monitoring with only a blood draw is a promising approach. Signal of the leukemia fingerprint is also detectable in urine, but at reduced levels and much more variable across patients. Conclusion: Our study shows the capability and utility of a personalized genomic residual disease detection method using MASQ to monitor patients and predict patient outcome. The high sensitivity and accurate quantitation of MASQ are demonstrated in a case study where relapse could be detected molecularly 7 months prior to clinical identification. Collection and analysis of a larger case series is ongoing.

Multicentric pilot study to standardize clinical whole exome sequencing (WES) for cancer patients

A growing number of druggable targets and national initiatives for precision oncology necessitate broad genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a positive percentage agreement (PPA) between 91 and 95% and a positive predictive value (PPV) between 82 and 95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Explanations for deviations included low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79–1, TMB: 0.97–0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.

Leptospira transcriptome sequencing using long-read technology reveals unannotated transcripts and potential polyadenylation of RNA molecules.

Leptospirosis, caused by the spirochete bacteria Leptospira, is an emerging zoonosis that causes life-threatening disease in humans and animals. However, a comprehensive understanding of leptospiral RNA profiles is limited. In this study, we sequenced and analyzed the transcriptome of multiple Leptospira strains using Oxford Nanopore Technologies' direct cDNA and direct RNA sequencing methods. We identified new operons, RNA molecules, and evidence of potential posttranscriptional polyadenylation in Leptospira transcriptomes. Some RNA molecules that had not been previously annotated could be potential sRNA or noncoding RNA molecules that play a role in gene expression regulation in Leptospira. Interestingly, the majority of the new RNA molecules identified in this study were not detected in the nonpathogenic Leptospira, suggesting potential virulence-related functions for these molecules. Overall, our study highlights the utility of Oxford Nanopore Technologies's sequencing in studying prokaryotic transcriptome profiles and offers a tool to improve our understanding of prokaryotic RNA landscapes and polyadenylation. Nonetheless, the findings from our study also warrant that the presence of homopolymers of adenine bases in the transcripts may interfere with the interpretation of bacterial transcriptome profiles. Carefully designed experiments are needed to explore the role of the features described in this study in Leptospira virulence and pathogenesis. IMPORTANCE Leptospirosis, caused by the spirochete bacteria Leptospira, is a zoonotic disease of humans and animals, accounting for over 1 million annual human cases and over 60,000 deaths. We have characterized operon transcriptional units, identified novel RNA coding regions, and reported evidence of potential posttranscriptional polyadenylation in the Leptospira transcriptomes for the first time using Oxford Nanopore Technology RNA sequencing protocols. The newly identified RNA coding regions and operon transcriptional units were detected only in the pathogenic Leptospira transcriptomes, suggesting their significance in virulence-related functions. This article integrates bioinformatics, infectious diseases, microbiology, molecular biology, veterinary sciences, and public health. Given the current knowledge gap in the regulation of leptospiral pathogenicity, our findings offer valuable insights to researchers studying leptospiral pathogenicity and provide both a basis and a tool for researchers focusing on prokaryotic molecular studies for the understanding of RNA compositions and prokaryotic polyadenylation for their organisms of interest.

Evaluation of next generation sequencing approaches for SARS-CoV-2

Within public health control strategies for SARS-CoV-2, whole genome sequencing (WGS) is essential for tracking viral spread and monitoring the emergence of variants which may impair the effectiveness of vaccines, diagnostic methods, and therapeutics. In this manuscript different strategies for SARS-CoV-2 WGS including metagenomic shotgun (SG), library enrichment by myBaits® Expert Virus-SARS-CoV-2 (Arbor Biosciences), nCoV-2019 sequencing protocol, ampliseq approach by Swift Amplicon® SARS-CoV-2 Panel kit (Swift Biosciences), and Illumina COVIDSeq Test (Illumina Inc.), were evaluated in order to identify the best approach in terms of results, labour, and costs. The analysis revealed that Illumina COVIDSeq Test (Illumina Inc.) is the best choice for a cost-effective, time-consuming production of consensus sequences.

Quantification of Poly(A) Tail Length and Terminal Modifications Using Direct RNA Sequencing.

Poly(A) tail metabolism is critical for various biological processes, including early embryogenesis and cell differentiation. While traditional biochemical methods to measure poly(A) tail length allow for the study of selected transcripts, the advent of long-read sequencing technologies enabled the development of simple and robust protocols to measure poly(A) tail length at the transcriptome level. Here, we describe a direct RNA sequencing protocol to capture poly(A) tail terminal additions based on the splint ligation of barcoded oligos compatible with terminal guanylation and uridylation. We cover how to prepare the libraries and perform the bioinformatics analysis to simultaneously determine the length of the transcripts' poly(A) tails and detect the presence of terminal guanylation and uridylation.

Restrander: rapid orientation and artefact removal for long-read cDNA data.

In transcriptomic analyses, it is helpful to keep track of the strand of the RNA molecules. However, the Oxford Nanopore long-read cDNA sequencing protocols generate reads that correspond to either the first or second-strand cDNA, therefore the strandedness of the initial transcript has to be inferred bioinformatically. Reverse transcription and PCR can also introduce artefacts which should be flagged in data pre-processing. Here we introduce Restrander, a lightning-fast and highly accurate tool for restranding and removing artefacts in long-read cDNA sequencing data. Thanks to its C++ implementation, Restrander was faster than Oxford Nanopore Technologies' existing tool Pychopper, and correctly restranded more reads due to its strategy of searching for polyA/T tails in addition to primer sequences from the reverse transcription and template-switch steps. We found that restranding improved the process of visualising and exploring data, and increased the number of novel isoforms discovered by bambu, particularly in regions where sense and anti-sense transcripts co-occur. The artefact detection implemented in Restrander quantifies reads lacking the correct 5' and 3' ends, a useful feature in quality control for library preparation. Restrander is pre-configured for all major cDNA protocols, and can be customised with user-defined primers. Restrander is available at https://github.com/mritchielab/restrander.

B-392 Standardization of Nucleic acid Extraction Method From Thyroid Fine Needle Aspiration (FNA) Cells on Stained and Fixed Slides for Next-Generation Sequencing Protocols

Abstract Background Thyroid Fine Needle Aspiration (FNA) cytopathology is the best method for histological classification of thyroid nodules. However, between 20%–30% of thyroid nodules show indeterminate classification and are often referred for surgery due to the malignancy risk. Post-surgical histological reports indicate that 84% of these cases are benign, highlighting a high rate of unnecessary surgeries. In this sense, molecular approaches using next-generation sequencing (NGS) have been useful to confirm benignity or malignancy. As important as the choice of a powerful methodology to evaluate different genes simultaneously such as NGS, is the standardization of nucleic acid extraction methods to allow sufficient quality recovery of these molecules. Pre-analytical steps involved in FNA slide preparation include fixation and stain methods that contribute to the low quality and poor recovery of nucleic acid, representing a great challenge to NGS analysis. Thus, this work aims to describe a standardization of nucleic acid extraction method from FNA slides for NGS protocols. Methodology Nucleic acid isolation from 30 FNA samples collected between 2016–2022 were performed by Trizol or RecoverAll kits, according to the manufacturer’s instructions (ThermoScientific). Initially, cells’ slides were counted in an optical microscope, considering cell clusters >10 (equivalent to 1 counter unit). Before extractions with Trizol, the slides were submerged in xylene for 15 min. For RecoverAll extractions, the varnished slides were previously exposed to dry ice for 5 min, while the slides containing coverslip were also exposed to dry ice and submerged in xylene for 15 min. In all cases, the slides were hydrated before scraping and then extracted. Subsequently, the extraction of whole slides and split slides was tested. To split slides, one-half of the slide was separated for RNA extraction, while the remaining half was for DNA extraction. Then, the DNA/RNA were quantified using the Qubit™ 4 Fluorometer equipment (ThermoScientific) and concentrated using Amicon® Ultra filters (Millipore). Results The DNA and RNA quantifications range from 0.0720–1.77 ng/µL and 0.508–7.43 ng/µL, respectively. Among samples, 12% (DNA) and 62.5% (RNA) presented concentration below the quantification limit (too low). Four samples extracted with Trizol did not obtain recovery of nucleic acid and this methodology was discontinued. The use of Amicon® filter improved all samples’ yield. The number of thyroid cell clusters was not proportional to the amount of DNA/RNA recovered. The split slides showed worst RNA/DNA recovery, being ideal to use at least two slides for each patient since co-extraction using the RecoverAll kit was not possible. Conclusion The isolations of nucleic acid from FNA slides are essential to performing subsequent molecular tests, such as NGS. However, specific protocols for nucleic acid extraction from FNA slides are hardly found in the literature. This work showed the combination of methodologies that allowed the scraping of material from FNA slides by exposure to dry ice in samples with varnish or coverslip. The nucleic acid recovery capacity described for this type of sample indicates a way of possibilities to improve the FNA extraction methodology.

Comparative analysis of bioinformatics tools to characterize SARS-CoV-2 subgenomic RNAs.

During the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), positive-sense genomic RNA and subgenomic RNAs (sgRNAs) are synthesized by a discontinuous process of transcription characterized by a template switch, regulated by transcription-regulating sequences (TRS). Although poorly known about makeup and dynamics of sgRNAs population and function of its constituents, next-generation sequencing approaches with the help of bioinformatics tools have made a significant contribution to expand the knowledge of sgRNAs in SARS-CoV-2. For this scope to date, Periscope, LeTRS, sgDI-tector, and CORONATATOR have been developed. However, limited number of studies are available to compare the performance of such tools. To this purpose, we compared Periscope, LeTRS, and sgDI-tector in the identification of canonical (c-) and noncanonical (nc-) sgRNA species in the data obtained with the Illumina ARTIC sequencing protocol applied to SARS-CoV-2-infected Caco-2 cells, sampled at different time points. The three software showed a high concordance rate in the identification and in the quantification of c-sgRNA, whereas more differences were observed in nc-sgRNA. Overall, LeTRS and sgDI-tector result to be adequate alternatives to Periscope to analyze Fastq data from sequencing platforms other than Nanopore.

Assessment and validation of enrichment and target capture approaches to improve Mycobacterium tuberculosis WGS from direct patient samples.

Within-host Mycobacterium tuberculosis (Mtb) diversity may detect antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here, we compared three sample pre-processing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Mtb-positive NALC-NaOH-treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I, or benzonase digestion. Levels of contaminating host DNA and target Mtb DNA were assessed by quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. The pre-treatment sample has a high host-to-target ratio of DNA (6,168 ± 1,638 host copies/ng to 212.3 ± 59.4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3,422 ± 2,162 host copies/ng to 11,721 ± 7,096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb target for all three pre-processing methods. Comparing the results of the benzonase sample sequenced both with and without enrichment, the percent of sequencing reads mapping to the Mtb increased to 90.95% from 1.18%. We demonstrate a low limit of detection for a new custom dsDNA Mtb target enrichment panel that has a favorable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

A paradigm shift in the prevention and diagnosis of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a widespread disease with only 50%-60% 5-year survival. Individuals with potentially malignant precursor lesions are at high risk. Survival could be increased by effective, affordable, and simple screening methods, along with a shift from incisional tissue biopsies to non-invasive brush biopsies for cytology diagnosis, which are easy to perform in primary care. Along with the explainable, fast, and objective artificial intelligence characterisation of cells through deep learning, an easy-to-use, rapid, and cost-effective methodology for finding high-risk lesions is achievable. The collection of cytology samples offers the further opportunity of explorative genomic analysis. Our prospective multicentre study of patients with leukoplakia yields a vast number of oral keratinocytes. In addition to cytopathological analysis, whole-slide imaging and the training of deep neural networks, samples are analysed according to a single-cell RNA sequencing protocol, enabling mapping of the entire keratinocyte transcriptome. Mapping the changes in the genetic profile, based on mRNA expression, facilitates the identification of biomarkers that predict cancer transformation. This position paper highlights non-invasive methods for identifying patients with oral mucosal lesions at risk of malignant transformation. Reliable non-invasive methods for screening at-risk individuals bring the early diagnosis of OSCC within reach. The use of biomarkers to decide on a targeted therapy is most likely to improve the outcome. With the large-scale collection of samples following patients over time, combined with genomic analysis and modern machine-learning-based approaches for finding patterns in data, this path holds great promise.

Imaging of Acute Abdominopelvic Pain in Pregnancy and Puerperium-Part II: Non-Obstetric Complications.

Emergency imaging in pregnancy and puerperium poses unique challenges both for clinicians and radiologists, requiring timely and accurate diagnosis. Delay in treatment may result in poor outcomes for both the patient and the foetus. Pregnant and puerperal patients may present in the emergency setting with acute abdominopelvic pain for various complications that can be broadly classified into obstetric and non-obstetric related diseases. Ultrasonography (US) is the primary diagnostic imaging test; however, it may be limited due to the patient's body habitus and the overlapping of bowel loops. Computed tomography (CT) carries exposure to ionising radiation to the foetus, but may be necessary in selected cases. Magnetic resonance imaging (MRI) is a valuable complement to US in the determination of the etiology of acute abdominal pain and can be used in most settings, allowing for the identification of a broad spectrum of pathologies with a limited protocol of sequences. In this second section, we review the common non-obstetric causes for acute abdominopelvic pain in pregnancy and post partum, offering a practical approach for diagnosis and pointing out the role of imaging methods (US, MRI, CT) with the respective imaging findings.

A Transcriptomic Analysis of Smoking-Induced Gene Expression Alterations in Coronary Artery Disease Patients.

Smoking is a well established risk factor for coronary artery disease (CAD). Despite this, there have been no previous studies investigating the effects of smoking on blood gene expression in CAD patients. This single-centre cross-sectional study was designed with clearly defined inclusion criteria to address this gap. We conducted a high-throughput approach using next generation sequencing analysis with a single-end sequencing protocol and a read length of 75-cycles. Sixty-one patients with a median age of 67 years (range: 28-88 years) were recruited, and only 44 subjects were included for further analyses. Our investigation revealed 120 differentially expressed genes (DEGs) between smokers and nonsmokers, with a fold change (FC) of ≥1.5 and a p-value < 0.05. Among these DEGs, 15 were upregulated and 105 were downregulated. Notably, when applying a more stringent adjusted FC ≥ 2.0, 31 DEGs (5 upregulated, annotated to immune response pathways, and 26 downregulated, involving oxygen and haem binding or activity, with FDR ≤ 0.03) remained statistically significant at an alpha level of <0.05. Our results illuminate the molecular mechanisms underlying CAD, fortifying existing epidemiological evidence. Of particular interest is the unexplored overexpression of RCAN3, TRAV4, and JCHAIN genes, which may hold promising implications for the involvement of these genes in CAD among smokers.

Limited Polymorphism in the Dihydrofolate Reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand

BackgroundThe 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of Disease Control in Thailand, suggesting a persistent infection. Little is known about dihydrofolate reductase (pkdhfr) and dihydropteroate synthase (pkdhps), putative antimalarial resistance markers for P. knowlesi. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating pkdhps mutations. The haplotype pattern of pkdhfr–pkdhps in Thai isolates was analyzed, and the effects of these pkdhps mutations were predicted by using a computer program. MethodsPkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein–ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates. ResultsFive nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein–ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr–pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates. ConclusionsA new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.

A versatile nuclei extraction protocol for single nucleus sequencing in non-model species-Optimization in various Atlantic salmon tissues.

The use of single cell sequencing technologies has exploded over recent years, and is now commonly used in many non-model species. Sequencing nuclei instead of whole cells has become increasingly popular, as it does not require the processing of samples immediately after collection. Here we present a highly effective nucleus isolation protocol that outperforms previously available method in challenging samples in a non-model specie. This protocol can be successfully applied to extract nuclei from a variety of tissues and species.

Analogous assembly mechanisms and functional guilds govern prokaryotic communities in mangrove ecosystems of China and South America.

As an important coastal "blue carbon sink," mangrove ecosystems contain microbial communities with an as-yet-unknown high species diversity. Exploring the assemblage and structure of sediment microbial communities therein can aid in a better understanding of their ecosystem functioning, such as carbon sequestration and other biogeochemical cycles in mangrove wetlands. However, compared to other biomes, the study of mangrove sediment microbiomes is limited, especially in diverse mangrove ecosystems at a large spatial scale, which may harbor microbial communities with distinct compositions and functioning. Here, we analyzed 380 sediment samples from 13 and 8 representative mangrove ecosystems, respectively, in China and South America and compared their microbial features. Although the microbial community compositions exhibited strong distinctions, the community assemblage in the two locations followed analogous patterns: the assemblages of the entire community, abundant taxa, rare taxa, and generalists were predominantly driven by stochastic processes with significant distance-decay patterns, while the assembly of specialists was more likely related to the behaviors of other organisms in or surrounding the mangrove ecosystems. In addition, co-occurrence and topological network analysis of mangrove sediment microbiomes underlined the dominance of sulfate-reducing prokaryotes in both the regions. Moreover, we found that more than 70% of the keystone and hub taxa were sulfate-reducing prokaryotes, implying their important roles in maintaining the linkage and stability of the mangrove sediment microbial communities. This study fills a gap in the large-scale analysis of microbiome features covering distantly located and diverse mangrove ecosystems. Here, we propose a suggestion to the Mangrove Microbiome Initiative that 16S rRNA sequencing protocols should be standardized with a unified primer to facilitate the global-scale analysis of mangrove microbiomes and further comparisons with the reference data sets from other biomes.IMPORTANCEMangrove wetlands are important ecosystems possessing valuable ecological functions for carbon storage, species diversity maintenance, and coastline stabilization. These functions are greatly driven or supported by microorganisms that make essential contributions to biogeochemical cycles in mangrove ecosystems. The mechanisms governing the microbial community assembly, structure, and functions are vital to microbial ecology but remain unclear. Moreover, studying these mechanisms of mangrove microbiomes at a large spatial scale can provide a more comprehensive insight into their universal features and can help untangle microbial interaction patterns and microbiome functions. In this study, we compared the mangrove microbiomes in a large spatial range and found that the assembly patterns and key functional guilds of the Chinese and South American mangrove microbiomes were analogous. The entire communities exhibited significant distance-decay patterns and were strongly governed by stochastic processes, while the assemblage of specialists may be merely associated with the behaviors of the organisms in mangrove ecosystems. Furthermore, our results highlight the dominance of sulfate-reducing prokaryotes in mangrove microbiomes and their key roles in maintaining the stability of community structure and functions.

Lessons learned: overcoming common challenges in reconstructing the SARS-CoV-2 genome from short-read sequencing data via CoVpipe2.

Accurate genome sequences form the basis for genomic surveillance programs, the added value of which was impressively demonstrated during the COVID-19 pandemic by tracing transmission chains, discovering new viral lineages and mutations, and assessing them for infectiousness and resistance to available treatments. Amplicon strategies employing Illumina sequencing have become widely established for variant detection and reference-based reconstruction of SARS-CoV-2 genomes, and are routine bioinformatics tasks. Yet, specific challenges arise when analyzing amplicon data, for example, when crucial and even lineage-determining mutations occur near primer sites. We present CoVpipe2, a bioinformatics workflow developed at the Public Health Institute of Germany to reconstruct SARS-CoV-2 genomes based on short-read sequencing data accurately. The decisive factor here is the reliable, accurate, and rapid reconstruction of genomes, considering the specifics of the used sequencing protocol. Besides fundamental tasks like quality control, mapping, variant calling, and consensus generation, we also implemented additional features to ease the detection of mixed samples and recombinants. We highlight common pitfalls in primer clipping, detecting heterozygote variants, and dealing with low-coverage regions and deletions. We introduce CoVpipe2 to address the above challenges and have compared and successfully validated the pipeline against selected publicly available benchmark datasets. CoVpipe2 features high usability, reproducibility, and a modular design that specifically addresses the characteristics of short-read amplicon protocols but can also be used for whole-genome short-read sequencing data. CoVpipe2 has seen multiple improvement cycles and is continuously maintained alongside frequently updated primer schemes and new developments in the scientific community. Our pipeline is easy to set up and use and can serve as a blueprint for other pathogens in the future due to its flexibility and modularity, providing a long-term perspective for continuous support. CoVpipe2 is written in Nextflow and is freely accessible from \href{https://github.com/rki-mf1/CoVpipe2}{github.com/rki-mf1/CoVpipe2} under the GPL3 license.

Sexually dimorphic effect of H-ferritin genetic manipulation on survival and tumor microenvironment in a mouse model of glioblastoma.

Iron plays a crucial role in various biological mechanisms and has been found to promote tumor growth. Recent research has shown that the H-ferritin (FTH1) protein, traditionally recognized as an essential iron storage protein, can transport iron to GBM cancer stem cells, reducing their invasion activity. Moreover, the binding of extracellular FTH1 to human GBM tissues, and brain iron delivery in general, has been found to have a sex bias. These observations raise questions, addressed in this study, about whether H-ferritin levels extrinsic to the tumor can affect tumor cell pathways and if this impact is sex-specific. To interrogate the role of systemic H-ferritin in GBM we introduce a mouse model in which H-ferritin levels are genetically manipulated. Mice that were genetically manipulated to be heterozygous for H-ferritin (Fth1+/-) gene expression were orthotopically implanted with a mouse GBM cell line (GL261). Littermate Fth1 +/+ mice were used as controls. The animals were evaluated for survival and the tumors were subjected to RNA sequencing protocols. We analyzed the resulting data utilizing the murine Microenvironment Cell Population (mMCP) method for in silico immune deconvolution. mMCP analysis estimates the abundance of tissue infiltrating immune and stromal populations based on cell-specific gene expression signatures. There was a clear sex bias in survival. Female Fth1+/- mice had significantly poorer survival than control females (Fth1+/+). The Fth1 genetic status did not affect survival in males. The mMCP analysis revealed a significant reduction in T cells and CD8 + T cell infiltration in the tumors of females with Fth1+/- background as compared to the Fth1+/+. Mast and fibroblast cell infiltration was increased in females and males with Fth1+/- background, respectively, compared to Fth1+/+ mice. Genetic manipulation of Fth1 which leads to reduced systemic levels of FTH1 protein had a sexually dimorphic impact on survival. Fth1 heterozygosity significantly worsened survival in females but did not affect survival in male GBMs. Furthermore, the genetic manipulation of Fth1 significantly affected tumor infiltration of T-cells, CD8 + T cells, fibroblasts, and mast cells in a sexually dimorphic manner. These results demonstrate a role for FTH1 and presumably iron status in establishing the tumor cellular landscape that ultimately impacts survival and further reveals a sex bias that may inform the population studies showing a sex effect on the prevalence of brain tumors.

Partial Genome Characterization of Novel Parapoxvirus in Horse, Finland.

We report a sequencing protocol and 121-kb poxvirus sequence from a clinical sample from a horse in Finland with dermatitis. Based on phylogenetic analyses, the virus is a novel parapoxvirus associated with a recent epidemic; previous data suggest zoonotic potential. Increased awareness of this virus and specific diagnostic protocols are needed.

A comparative investigation of single nucleotide variant calling for a personal non-Caucasian sequencing sample.

Dropping cost and increasing clinical application of whole genome sequencing (WGS) lead a necessity of efficient (accurate and rapid) variant calling procedures from a personal WGS data (n = 1). A number of variant calling pipelines have been introduced utilizing the human genome reference GRCh38 as a reference and a benchmark dataset called 'NA12878', which are both 'standard' but limited ethnic origin. Considering the nature of variant calling algorithms and recent updates in sequencing protocol, however, it is necessary to revisit the efficiency of the current best pipelines for a personal WGS data from diverse ethnicity. We discuss the most efficient practices for variant calling of a personal WGS reads, with a particular emphasis on whether (1) ethnic match or mismatch between the reference genome and a WGS data produces a distinct result and more importantly (2) there is an ethnic-specific optimal workflow. Here, we generate an appropriate WGS data, DNA array, and sufficient number of Sanger validated variants from a single Korean subject to perform such a comprehensive comparison. We applied this WGS reads and the 'NA12878' reads to 8 different variant calling pipelines with 2 different reference genomes (GRCh38 and KOREF, a Korean reference genome) to which the WGS reads from different ethnic origins are aligned. We evaluated the performance of the pipelines with the matched array genotype data and Sanger sequencing validation and demonstrated that: regardless to the ethnic match/mismatch (1) Novoalign-GATK4 showed the most efficient performance with the exceptional calls in MHC region; (2) the overall performance was better with GRCh38, while a significant difference in recall was observed. In addition, we found it is largely reduced computing cost maintaining performance to remove 'markduplication' step with PCR-free WGS data. For variant calling of a personal PCR-free WGS data, regardless of ethnicity consideration, we recommend the use of the Novoalign + GATK4 with GRCh38 and without 'markduplication'.

Unveiling the Protein Components of the Secretory-Venom Gland and Venom of the Scorpion Centruroides possanii (Buthidae) through Omic Technologies.

Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.