High-throughput DNA Sequencing Research Articles

Simultaneous measurement of the antibody responses against SARS-CoV-2 and its multiple variants by a phage display mediated immuno-multiplex quantitative PCR-based assay.

To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen–antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.

Comparative Analysis of Bacillariophyceae Chloroplast Genomes Uncovers Extensive Genome Rearrangements Associated with Speciation.

The Bacillariophyceae is a species-rich, ecologically significant class of Bacillariophyta. Despite their critical importance in marine ecosystems as primary producers and in the development of harmful algal blooms (HABs), taxonomic research on Bacillariophyceae species has been hindered because of their limited morphological features, plasticity of morphologies, and the low resolution of common molecular markers. Hence molecular markers with improved resolution are urgently needed. Organelle genomes, which can be constructed efficiently with the recent development of high throughput DNA sequencing technologies and the advancement of bioinformatics tools, have been proposed as super barcodes for their higher resolution for distinguishing different species and intra-species genomic variations. In this study, we tested the value of full-length chloroplast genomes (cpDNAs) as super barcodes for distinguishing diatom species, by constructing cpDNAs of 11 strains of the class Bacillariophyceae, including Nitzschia ovalis, Nitzschia traheaformis, Cylindrotheca spp., Psammodictyon constrictum, Bacillaria paxillifer, two strains of Haslea tsukamotoi, Haslea avium, Navicula arenaria, and Pleurosigma sp. Comparative analysis of cpDNAs revealed that cpDNAs were not only adequate for resolving different species, but also for enabling recognition of high levels of genome rearrangements between cpDNAs of different species, especially for species of the genera Nitzschia, Cylindrotheca, Navicula and Haslea. Additionally, comparative analysis suggested that the positioning of species in the genus Haslea should be transferred to the genus Navicula. Chloroplast genome-based evolutionary analysis suggested that the Bacillariophyceae species first appeared during the Cretaceous period and the diversity of species rose after the mass extinction about 65 Mya. This study highlighted the value of cpDNAs in research on the biodiversity and evolution of Bacillariophyceae species, and, with the construction of more cpDNAs representing additional genera, deeper insight into the biodiversity and evolutionary relationships of Bacillariophyceae species will be gained.

A Case Study for the Recovery of Authentic Microbial Ancient DNA from Soil Samples.

High Throughput DNA Sequencing (HTS) revolutionized the field of paleomicrobiology, leading to an explosive growth of microbial ancient DNA (aDNA) studies, especially from environmental samples. However, aDNA studies that examine environmental microbes routinely fail to authenticate aDNA, examine laboratory and environmental contamination, and control for biases introduced during sample processing. Here, we surveyed the available literature for environmental aDNA projects—from sample collection to data analysis—and assessed previous methodologies and approaches used in the published microbial aDNA studies. We then integrated these concepts into a case study, using shotgun metagenomics to examine methodological, technical, and analytical biases during an environmental aDNA study of soil microbes. Specifically, we compared the impact of five DNA extraction methods and eight bioinformatic pipelines on the recovery of microbial aDNA information in soil cores from extreme environments. Our results show that silica-based methods optimized for aDNA research recovered significantly more damaged and shorter reads (<100 bp) than a commercial kit or a phenol–chloroform method. Additionally, we described a stringent pipeline for data preprocessing, efficiently decreasing the representation of low-complexity and duplicated reads in our datasets and downstream analyses, reducing analytical biases in taxonomic classification.

Cyclobutane Pyrimidine Dimer Hyperhotspots as Sensitive Indicators of Keratinocyte UV Exposure†.

The dominant DNA damage generated by UV exposure is the cyclobutane pyrimidine dimer (CPD), which alters skin cell physiology and induces cell death and mutation. Genome-wide nucleotide-resolution analysis of CPDs in melanocytes and fibroblasts has identified "CPD hyperhotspots", pyrimidine-pyrimidine sites hundreds of fold more susceptible to the generation of CPDs than the genomic average. Identifying hyperhotspots in keratinocytes could enable measuring individual past UV exposure in small skin samples and predicting future skin cancer risk. We therefore exposed neonatal human epidermal keratinocytes to narrowband UVB and quantified CPDs using the adductSeq high-throughput DNA sequencing method. Keratinocytes contained thousands of CPD hyperhotspots, with a UVB-sensitivity up to 550 fold greater than the genomic average. As with melanocytes, the most sensitive sites were located in promoter regions at ETS-family transcription factor binding sequence motifs, near RNA processing genes. Moreover, they lay at sequence motifs bound to ETS1 in CpG islands. These genes were specifically upregulated in skin and the CPD hyperhotspots were mutated in a fraction of keratinocyte cancers. Crucially for their biological importance and practical application, CPD hyperhotspot locations and UV-sensitivity ranking demonstrated high reproducibility across experiments and across skin donors. CPD hyperhotspots are therefore sensitive indicators of UV exposure.

Plastisphere in lake waters: Microbial diversity, biofilm structure, and potential implications for freshwater ecosystems

Once dispersed in water, microplastic (MP) particles are rapidly colonised by aquatic microbes, which can adhere and grow onto solid surfaces in the form of biofilms. This study provides new insights on microbial diversity and biofilm structure of plastisphere in lake waters. By combining Fourier Confocal Laser Scanning Microscopy (CLSM), Transform Infrared Spectroscopy (FT-IR) and high-throughput DNA sequencing, we investigated the microbial colonization patterns on floating MPs and, for the first time, the occurrence of eukaryotic core members and their possible relations with biofilm-forming bacterial taxa within the plastisphere of four different lakes. Through PCR-based methods (qPCR, LAMP-PCR), we also evaluated the role of lake plastisphere as long-term dispersal vectors of potentially harmful organisms (including pathogens) and antibiotic resistance genes (ARGs) in freshwater ecosystems. Consistent variation patterns of the microbial community composition occurred between water and among the plastisphere samples of the different lakes. The eukaryotic core microbiome was mainly composed by typical freshwater biofilm colonizers, such as diatoms (Pennales, Bacillariophyceaea) and green algae (Chlorophyceae), which interact with eukaryotic and prokaryotic microbes of different trophic levels. Results also showed that MPs are suitable vectors of biofilm-forming opportunistic pathogens and a hotspot for horizontal gene transfer, likely facilitating antibiotic resistance spread in the environments.

Sapporo: A workflow execution service that encourages the reuse of workflows in various languages in bioinformatics

The increased demand for efficient computation in data analysis encourages researchers in biomedical science to use workflow systems. Workflow systems, or so-called workflow languages, are used for the description and execution of a set of data analysis steps. Workflow systems increase the productivity of researchers, specifically in fields that use high-throughput DNA sequencing applications, where scalable computation is required. As systems have improved the portability of data analysis workflows, research communities are able to share workflows to reduce the cost of building ordinary analysis procedures. However, having multiple workflow systems in a research field has resulted in the distribution of efforts across different workflow system communities. As each workflow system has its unique characteristics, it is not feasible to learn every single system in order to use publicly shared workflows. Thus, we developed Sapporo, an application to provide a unified layer of workflow execution upon the differences of various workflow systems. Sapporo has two components: an application programming interface (API) that receives the request of a workflow run and a browser-based client for the API. The API follows the Workflow Execution Service API standard proposed by the Global Alliance for Genomics and Health. The current implementation supports the execution of workflows in four languages: Common Workflow Language, Workflow Description Language, Snakemake, and Nextflow. With its extensible and scalable design, Sapporo can support the research community in utilizing valuable resources for data analysis.

The genome and diet of a 35,000-year-old Canis lupus specimen from the Paleolithic painted cave, Chauvet-Pont d'Arc, France.

The Chauvet-Pont-d'Arc Cave (Ardèche, France) contains some of the oldest Paleolithic paintings recorded to date, as well as thousands of bones of the extinct cave bear, and some remains and footprints of other animals. As part of the interdisciplinary research project devoted to this reference cave site, we analyzed a coprolite collected within the deep cave. AMS radiocarbon dating of bone fragments from the coprolite yielded an age of 30,450 ± 550 RC yr. BP (AAR-19656; 36,150-34,000 cal BP), similar to ages assigned to Paleolithic artwork and cave bear remains from the same cave sector. Using high-throughput shotgun DNA sequencing, we demonstrated a high abundance of canid DNA and lesser amounts of DNA from the extinct cave bear. We interpret the sample as feces from a canid that had consumed cave bear tissue. The high amount of canid DNA allowed us to reconstruct a complete canid mitochondrial genome sequence (average coverage: 83×) belonging to a deeply divergent clade of extinct mitochondrial wolf lineages that are most closely related to coeval (~35 ka) Belgian wolves. Analysis of the nuclear genome yielded a similar coverage for the X chromosome (2.4×) and the autosomes (range: 2.3-3.2×), indicating that the Chauvet canid was a female. Comparing the relationship of the nuclear genome of this specimen with that of a variety of canids, we found it more closely related to gray wolves' genomes than to other wild canid or dog genomes, especially wolf genomes from Europe and the Middle East. We conclude that the coprolite is feces from an animal within an extinct wolf lineage. The consumption of cave bear by this wolf likely explains its intrusion into the dark cave sectors and sheds new light on the paleoecology of a major cave site.

Complete genome sequencing and investigation on the fiber-degrading potential of Bacillus amyloliquefaciens strain TL106 from the tibetan pig

BackgroundCellulolytic microorganisms are considered a key player in the degradation of feed fiber. These microorganisms can be isolated from various resources, such as animal gut, plant surfaces, soil and oceans. A new strain of Bacillus amyloliquefaciens, TL106, was isolated from faeces of a healthy Tibetan pigs. This strain can produce cellulase and shows strong antimicrobial activity in mice. Thus, in this study, to better understand the strain of B. amyloliquefaciens TL106 on degradation of cellulose, the genome of the strain TL106 was completely sequenced and analyzed. In addition, we also explored the cellulose degradation ability of strain TL106 in vitro.ResultsTL106 was completely sequenced with the third generation high-throughput DNA sequencing. In vitro analysis with enzymatic hydrolysis identified the activity of cellulose degradation. TL106 consisted of one circular chromosome with 3,980,960 bp and one plasmid with 16,916 bp, the genome total length was 3.99 Mb and total of 4,130 genes were predicted. Several genes of cellulases and hemicellulase were blasted in Genbank, including β-glucosidase, endoglucanase, ß-glucanase and xylanase genes. Additionally, the activities of amylase (20.25 U/mL), cellulase (20.86 U/mL), xylanase (39.71 U/mL) and β-glucanase (36.13 U/mL) in the fermentation supernatant of strain TL106 were higher. In the study of degradation characteristics, we found that strain TL106 had a better degradation effect on crude fiber, neutral detergent fiber, acid detergent fiber, starch, arabinoxylan and β-glucan of wheat and highland barley .ConclusionsThe genome of B. amyloliquefaciens TL106 contained several genes of cellulases and hemicellulases, can produce carbohydrate-active enzymes, amylase, cellulase, xylanase and β-glucanase. The supernatant of fermented had activities of strain TL106. It could degrade the fiber fraction and non-starch polysaccharides (arabinoxylans and β-glucan) of wheat and highland barley. The present study demonstrated that the degradation activity of TL106 to crude fiber which can potentially be applied as a feed additive to potentiate the digestion of plant feed by monogastric animals.

Mutational Signature and Integrative Genomic Analysis of Human Papillomavirus-Associated Penile Squamous Cell Carcinomas from Latin American Patients.

Simple SummaryDNA sequencing has been crucial to comprehending cancer mutational patterns, leading to the identification of driver genes and altered signaling pathways. Thus, identifying new pathogenic variants and their impact on tumor onset, progression, and treatment response has fueled tumor biology research. Here, we present novel findings addressing the first whole-exome sequencing (WES) of human papillomavirus (HPV)-associated penile squamous cell carcinoma (PSCC) from Latin Americans and its association with pathogenesis. We also compared the molecular profile of the tumors to that of three previous studies from populations with different genetic and socioeconomic backgrounds, the majority of which was HPV-negative. We describe the most altered genes and the main pathogenic variants found in the Latin Americans, ten of which are exclusive to our study sample. The data allowed us to identify molecular pathways and druggable targets with potential treatment value for this still-neglected HPV-associated carcinoma.High-throughput DNA sequencing has allowed for the identification of genomic alterations and their impact on tumor development, progression, and therapeutic responses. In PSCC, for which the incidence has progressively increased worldwide, there are still limited data on the molecular mechanisms involved in the disease pathogenesis. In this study, we characterized the mutational signature of 30 human papillomavirus (HPV)-associated PSCC cases from Latin Americans, using whole-exome sequencing. Copy number variations (CNVs) were also identified and compared to previous array-generated data. Enrichment analyses were performed to reveal disrupted pathways and to identify alterations mapped to HPV integration sites (HPVis) and miRNA–mRNA hybridization regions. Among the most frequently mutated genes were NOTCH1, TERT, TTN, FAT1, TP53, CDKN2A, RYR2, CASP8, FBXW7, HMCN2, and ITGA8. Of note, 92% of these altered genes were localized at HPVis. We also found mutations in ten novel genes (KMT2C, SMARCA4, PTPRB, AJUBA, CR1, KMT2D, NBEA, FAM135B, GTF2I, and CIC), thus increasing our understanding of the potential HPV-disrupted pathways. Therefore, our study reveals innovative targets with potential therapeutic benefits for HPV-associated PSCCs. The CNV analysis by sequencing (CNV-seq) revealed five cancer-associated genes as the most frequent with gains (NOTCH1, MYC, NUMA1, PLAG1, and RAD21), while 30% of the tumors showed SMARCA4 with loss. Additionally, four cancer-associated genes (CARD11, CSMD3, KDR, and TLX3) carried untranslated regions (UTRs) variants, which may impact gene regulation by affecting the miRNAs hybridization regions. Altogether, these data contribute to the characterization of the mutational spectrum and its impact on cellular signaling pathways in PSCC, thus reinforcing the pivotal role of HPV infection in the molecular pathogenesis of these tumors.

Conjunctival Intraepithelial Lymphocytes, Lacrimal Cytokines and Ocular Commensal Microbiota: Analysis of the Three Main Players in Allergic Conjunctivitis.

Conjunctival intraepithelial lymphocytes, tear soluble molecules and commensal microbiota have important roles in the ocular mucosal immune response in healthy and diseased subjects. For the purpose of this study, the cellular and microbial populations of the conjunctiva and the lacrimal soluble molecules were analyzed to find the main biomarkers in allergic conjunctivitis. A total of 35 healthy subjects, 28 subjects with seasonal allergic conjunctivitis and 32 subjects with perennial allergic conjunctivitis were recruited to obtain peripheral blood, conjunctival brush cytology, tear fluid and microbiota samples. Flow cytometry for lymphocytes, multiplex bead assays for cytokines and high-throughput DNA sequencing for microbiome analysis were used. For perennial allergic conjunctivitis, an increased proportion of Th2 and NKT lymphocytes was found, while CD3+TCRγδ+ lymphocytes and double negative MAIT cells were decreased. In contrast, seasonal allergic conjunctivitis was distinguished by an increase in Th17 and Th22 cell proportions, while the Th1 cell proportion decreased. Among tear fluid, the vast majority of pro-inflammatory cytokines (especially Th2 and Th17 cytokines) in perennial allergies and MMP-9 together with IgA in seasonal allergies were increased. In contrast, TGF-β2 was decreased in both forms of conjunctivitis. Finally, fungal (Malassezia species) and bacterial (Kocuria and Propionobacterium acnes species) colonization were observed in the perennial allergic conjunctivitis group. These results provide the basis for the development of a disease profile for perennial allergic conjunctivitis and open the door to new therapeutic and diagnostic strategies.

Suppression of Allergic Asthma by Loss of Function of Miz1-mediated Th1 Skewing.

Asthma is the most prevalent chronic respiratory disease worldwide. There is currently no cure, and it remains an important cause of morbidity and mortality. Here we report that lung-specific loss of function of the transcription factor Miz1 (c-Myc-interacting zinc finger protein-1) upregulates the pro-T-helper cell type 1 cytokine IL-12. Upregulation of IL-12 in turn stimulates a Th1 response, thereby counteracting T-helper cell type 2 response and preventing the allergic response in mouse models of house dust mite- and OVA (ovalbumin)-induced asthma. Using transgenic mice expressing Cre under a cell-specific promoter, we demonstrate that Miz1 acts in lung epithelial cells and dendritic cells in asthma. Chromatin immunoprecipitation coupled with high-throughput DNA sequencing or quantitative PCR reveals the binding of Miz1 on the Il12 promoter indicating direct repression of IL-12 by Miz1. In addition, HDAC1 (histone deacetylase 1) is recruited to the Il12 promoter in a Miz1-depdenent manner, suggesting epigenetic repression of Il12 by Miz1. Furthermore, Miz1 is upregulated in the lungs of asthmatic mice. Our data together suggest that Miz1 is upregulated during asthma, which in turn promotes asthma pathogenesis by preventing Th1 skewing through the transcriptional repression of IL-12.

A probable cis-acting genetic modifier of Huntington disease frequent in individuals with African ancestry.

SummaryHuntington disease (HD)is a dominantly inherited neurodegenerative disorder caused by the expansion of a polyglutamine encoding CAG repeat in the huntingtin gene. Recently, it has been established that disease severity in HD is best predicted by the number of pure CAG repeats rather than total glutamines encoded. Along with uncovering DNA repair gene variants as trans-acting modifiers of HD severity, these data reveal somatic expansion of the CAG repeat as a key driver of HD onset. Using high-throughput DNA sequencing, we have determined the precise sequence and somatic expansion profiles of the HTT repeat tract of 68 HD-affected and 158 HD-unaffected African ancestry individuals. A high level of HTT repeat sequence diversity was observed, with three likely African-specific alleles identified. In the most common disease allele (30 out of 68), the typical proline-encoding CCGCCA sequence was absent. This CCGCCA-loss disease allele was associated with an earlier age of diagnosis of approximately 7.1 years and occurred exclusively on haplotype B2. Although somatic expansion was associated with an earlier age of diagnosis in the study overall, the CCGCCA-loss disease allele displayed reduced somatic expansion relative to the typical HTT expansions in blood DNA. We propose that the CCGCCA loss occurring on haplotype B2 is an African cis-acting modifier that appears to alter disease diagnosis of HD through a mechanism that is not driven by somatic expansion. The assessment of a group of individuals from an understudied population has highlighted population-specific differences that emphasize the importance of studying genetically diverse populations in the context of disease.

Evolutionary innovations of human cerebral cortex viewed through the lens of high-throughput sequencing.

Humans had acquired a tremendously enlarged cerebral cortex containing a huge quantity and variety of cells during evolution. Such evolutionary uniqueness offers a neural basis of our cognitive innovation and human-specific features of neurodevelopmental and psychiatric disorders. Since human brain is hardly examined in vivo with experimental approaches commonly applied on animal models, the recent advancement of sequencing technologies offers an indispensable viewpoint of human brain anatomy and development. This review introduces the recent findings on the unique features in the adult and the characteristic developmental processes of the human cerebral cortex, based on high-throughput DNA sequencing technologies.

Ionic Liquid Decelerates Single-Stranded DNA Transport through Molybdenum Disulfide Nanopores.

Nanopores in two-dimensional (2D) materials have emerged to offer in principle necessary spatial resolution for high-throughput DNA sequencing. However, their fidelity is severely limited by the fast DNA translocation. A recent experiment indicates that introducing ionic liquids could slow down DNA translocation in a MoS2 nanopore. However, the corresponding in-depth molecular mechanism underlying the experimental findings is not fully understood, which is crucial for the future improvement of rational DNA translocation control. Here, we computationally investigate and then experimentally identify the effect of BmimCl ionic liquid on the retardation of ssDNA translocation through a single-layer MoS2 nanopore. Our all-atom molecular dynamics simulations demonstrate that the strong interaction between Bmim+ and ssDNA offers a considerable dragging force to decelerate the electrophoretic motion of ssDNA in the BmimCl solution. Moreover, we show that Bmim+ ions exhibit preferential binding on the sulfur edges of the nanopore. These Bmim+ in the pore region can not only act as a steric blockage but also form π-π stackings with nucleobases, which provide a further restriction on the ssDNA motion. Therefore, our molecular dynamics simulation investigations deepen the understanding of the critical role of ionic liquid in DNA translocation through a nanopore from a molecular landscape, which may benefit practical implementations of ionic liquids in nanopore sequencing.

Clinical manifestations of clonal hematopoiesis: What has SF3B1-mutant MDS taught us?

Large scale high-throughput DNA sequencing studies have identified clonal hematopoiesis (CH) as a clinical phenomenon characterized by a disproportionately large clonal population in the hematopoietic system with a shared mutational background. CH originates through mutations in hematopoietic stem and progenitor cells (HSPCs) which provide a proliferative advantage over unmutated HSPCs and has been characterized as a risk factor for myeloid neoplasm (MN) development. Large population studies found that CH is an age-related event which is commonly found in association with milder phenotypes such as cytopenia, mild monocytosis, intravascular hemolysis, or chronic inflammation. More importantly, the vast majority of individuals with CH are asymptomatic and healthy people of advanced age, where the impact of CH is thus considered to be of indeterminate potential (CHIP). These conditions are sometimes referred to as benign to facilitate distinction from overt MN but, despite this definition, may still result in severe illness, reduced overall survival, and increased risk of hematologic neoplasms development and all-cause mortality. The purpose of this review is to describe clinical conditions associated with CH, the clinical significance of CH-related clinical phenotypes, and the determinants of progression from CH to overt MN following the paradigmatic example of SF3B1-driven CH.

Revealing the Microbiome of Four Different Thermal Springs in Turkey with Environmental DNA Metabarcoding.

Simple SummaryThe physicochemical conditions of thermal springs are one of the most significant barriers for detecting microbial life. According to the findings of various studies, high-throughput DNA sequencing technology can be utilized to perform more precise and detailed microbiome assessments. The main goal of this paper was to determine the microbiome in a thermal spring by metabarcoding environmental DNA obtained from four different sources and revealing how temperature and chemical composition affect the microbiome. This research also aimed to gather information that will aid in determining the best gene region and bioinformatic pipeline. The findings revealed a link between four different thermal springs’ physicochemical parameters and microbial composition and we found various manipulable steps in this study. This research is also first comprehensive thermal spring metabarcoding study conducted in Turkey.One of the most significant challenges for detecting microbial life in thermal springs by conventional techniques such as culturing is these places’ physicochemical (temperature, heavy metal content, pH, etc.) conditions. Data from several studies suggest that high-throughput DNA sequencing technologies can be used to perform more accurate and detailed microbiome analyses. The primary aim of this paper was to determine the microbiome in the thermal source by metabarcoding environmental DNA isolated from four different sources and reveal the reflection of differences caused by temperature and chemical content on the microbiome. DNA was extracted from water filtered with enclosed filters and using the Illumina high-throughput sequencing platform, V3 and V4 regions of the 16S rRNA gene were sequenced. The results showed a correlation between physicochemical conditions and microorganism composition of four different thermal springs. Springs with extremely high temperature (89–90 °C) were dominated by hyperthermophiles such as Hydrogenobacter and Thermus, while a spring with a high temperature (52 °C) was dominated by thermophiles such as Thermoanaerobaculum and Desulfurispora, and a spring with a low temperature (26 °C) and high salinity was dominated by halophiles and sulfur-oxidizers such as Hydrogenovibrio and Sulfirimonas. With this research, we observed many manipulable steps according to the work of interest. This study sought to obtain data that will help decide the right gene region and choose the optimal bioinformatic pipeline.

Cell-Free DNA for Genomic Analysis in Primary Mediastinal Large B-Cell Lymphoma.

High-throughput sequencing of cell-free DNA (cfDNA) has emerged as a promising noninvasive approach in lymphomas, being particularly useful when a biopsy specimen is not available for molecular analysis, as it frequently occurs in primary mediastinal large B-cell lymphoma (PMBL). We used cfDNA for genomic characterization in 20 PMBL patients by means of a custom NGS panel for gene mutations and low-pass whole-genome sequencing (WGS) for copy number analysis (CNA) in a real-life setting. Appropriate cfDNA to perform the analyses was obtained in 18/20 cases. The sensitivity of cfDNA to detect the mutations present in paired FFPE samples was 69% (95% CI: 60–78%). The mutational landscape found in cfDNA samples was highly consistent with that of the tissue, with the most frequently mutated genes being B2M (61%), SOCS1 (61%), GNA13 (44%), STAT6 (44%), NFKBIA (39%), ITPKB (33%), and NFKBIE (33%). Overall, we observed a 75% concordance to detect CNA gains/losses between DNA microarray and low-pass WGS. The sensitivity of low-pass WGS was remarkably higher for clonal CNA (18/20, 90%) compared to subclonal alterations identified by DNA microarray. No significant associations between cfDNA amount and tumor burden or outcome were found. cfDNA is an excellent alternative source for the accurate genetic characterization of PMBL cases.

Sparse and skew hashing of K-mers.

MotivationA dictionary of k-mers is a data structure that stores a set of n distinct k-mers and supports membership queries. This data structure is at the hearth of many important tasks in computational biology. High-throughput sequencing of DNA can produce very large k-mer sets, in the size of billions of strings—in such cases, the memory consumption and query efficiency of the data structure is a concrete challenge.ResultsTo tackle this problem, we describe a compressed and associative dictionary for k-mers, that is: a data structure where strings are represented in compact form and each of them is associated to a unique integer identifier in the range . We show that some statistical properties of k-mer minimizers can be exploited by minimal perfect hashing to substantially improve the space/time trade-off of the dictionary compared to the best-known solutions.Availability and implementation https://github.com/jermp/sshash.Supplementary information Supplementary data are available at Bioinformatics online.

Applying convolutional neural networks to speed up environmental DNA annotation in a highly diverse ecosystem

High-throughput DNA sequencing is becoming an increasingly important tool to monitor and better understand biodiversity responses to environmental changes in a standardized and reproducible way. Environmental DNA (eDNA) from organisms can be captured in ecosystem samples and sequenced using metabarcoding, but processing large volumes of eDNA data and annotating sequences to recognized taxa remains computationally expensive. Speed and accuracy are two major bottlenecks in this critical step. Here, we evaluated the ability of convolutional neural networks (CNNs) to process short eDNA sequences and associate them with taxonomic labels. Using a unique eDNA data set collected in highly diverse Tropical South America, we compared the speed and accuracy of CNNs with that of a well-known bioinformatic pipeline (OBITools) in processing a small region (60 bp) of the 12S ribosomal DNA targeting freshwater fishes. We found that the taxonomic labels from the CNNs were comparable to those from OBITools, with high correlation levels for the composition of the regional fish fauna. The CNNs enabled the processing of raw fastq files at a rate of approximately 1 million sequences per minute, which was about 150 times faster than with OBITools. Given the good performance of CNNs in the highly diverse ecosystem considered here, the development of more elaborate CNNs promises fast deployment for future biodiversity inventories using eDNA.

Abstract 5791: Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples

Abstract Introduction We developed a unique panel of 54 target genes dedicated to hematological malignancies (HematoPanel) and screened 1,108 clinical samples. We also performed transcriptomic analysis and evaluated its relevance for patients with acute lymphoblastic leukemia (ALL). Methods DNA and RNA were extracted from cell pellets with the Maxwell® RSC (Promega®). Libraries for targeted high-throughput DNA sequencing (HTS) were generated in duplicate from 100 ng with the Advanta™ NGS Library Prep Assay kit using the LP 48.48 IFC (integrated fluidic circuit) on a Juno™ and sequenced on the NextSeq™ 550 (Illumina®) to a median depth of coverage of approximately 1,200X per sample. Transcriptomic libraries were generated from 50 ng RNA with the Advanta RNA-Seq XT NGS Library Prep Kit using the LP 48.Atlas™ IFC on Juno and sequenced on the NextSeq 550. A bioinformatics pipeline was developed for fusion transcript detection. Differential gene expression was analyzed with the DESeq2 package after STAR alignment. Results In 2020 we sequenced 1,108 consecutive samples in routine practice with HematoPanel. The main indications were the suspicion of myeloproliferative neoplasms (32%), the evaluation of chronic lymphocytic leukemia (23%), and the diagnostic workup of cytopenias (15%). We detected 1,306 mutations in 654 patients (59%) (1 to 8 mutations per sample). Eight genes (TET2, SF3B1, TP53, DNMT3A, ASXL1, JAK2, SRSF2 and MYD88) were reported as mutated for more than 50 patients. We sequenced 22 samples including the MOLT-4 cell line, healthy donor samples, and 16 ALL samples for transcriptomic evaluation. From ALL samples, we detected 5 fusion transcripts previously identified by specific PCR (1 BCR-ABL, 2 ETV6-RUNX1, 1 KMT2A-AFF1, 1 TCF3-PBX1). Moreover, we detected 4 other fusion transcripts for 3 patients (EBF1-PDGFRB, FGFR1-MYO18A, P2RY8-CRLF2, and PAX5-NOL4L). Interestingly, these patients clustered with the BCR-ABL positive sample based on their expression profile. It confirms the diagnosis of Phi-like ALL and thus offers a therapeutic target for these patients. Conclusion Microfluidics-based library prep using Juno offers a cost-effective strategy and straightforward workflows for both targeted HTS and RNA sequencing. We validated its use for hematological malignancy evaluation in routine practice. Citation Format: Cédric Pastoret, Amyra Aliouat, Marie-Laure Boulland, Florent Denoual, Anne Desmares, Marie De Tayrac, Thierry Fest, Celeste Letellier. Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5791.