Sequencing Libraries Research Articles

RiboSeq.Org: an integrated suite of resources for ribosome profiling data analysis and visualization

Abstract Ribosome profiling (Ribo-Seq) has revolutionised our understanding of translation, but the increasing complexity and volume of Ribo-Seq data present challenges for its reuse. Here, we formally introduce RiboSeq.Org, an integrated suite of resources designed to facilitate Ribo-Seq data analysis and visualisation within a web browser. RiboSeq.Org comprises several interconnected tools: GWIPS-viz for genome-wide visualisation, Trips-Viz for transcriptome-centric analysis, RiboGalaxy for data processing and the newly developed RiboSeq data portal (RDP) for centralised dataset identification and access. The RDP currently hosts preprocessed datasets corresponding to 14840 sequence libraries (samples) from 969 studies across 96 species, in various file formats along with standardised metadata. RiboSeq.Org addresses key challenges in Ribo-Seq data reuse through standardised sample preprocessing, semi-automated metadata curation and programmatic information access via a REST API and command-line utilities. RiboSeq.Org enhances the accessibility and utility of public Ribo-Seq data, enabling researchers to gain new insights into translational regulation and protein synthesis across diverse organisms and conditions. By providing these integrated, user-friendly resources, RiboSeq.Org aims to lower the barrier to reproducible research in the field of translatomics and promote more efficient utilisation of the wealth of available Ribo-Seq data.

Abstract B057: Base-error model (BEM) for improved detection of ctDNA in lymph samples of HPV-negative head and neck cancer patients

Abstract Introduction: Previously we demonstrated that ctDNA present in lymphatic exudate collected via surgical drains (“lymph”) outperformed plasma for detecting minimal residual disease (MRD) in head and neck squamous cell carcinoma (HNSCC) patients through a targeted sequencing (TS) approach. However, artifacts introduced during library preparation and sequencing remain challenges to sensitive low allelic fraction mutation detection. To enhance the performance of the TS approach, we characterized the background noise in lymph samples and developed a base-error model (BEM) that reduces sequencing artifacts and maximizes ctDNA signal. Methods: Lymph and blood were collected from 44 HPV-negative HNSCC patients postoperatively at 24 hours along with resected tumor. Cell-free DNA was extracted from lymph and sequenced using a custom TS panel with unique molecular identifiers (UMI). Somatic mutations were identified by tumor and blood exome sequencing (200x). Five patients had <2 mutations in tumor and were excluded. Two patients were censored due to lack of clinical data, yielding 17 patients with disease recurrence (REC) and 20 with no evidence of disease (NED) with >1 year of follow-up. Tumor-specific variants were force-called in lymph using a custom pipeline. A BEM of each tumor variant was built using a series of high-quality lymph reference samples to quantify the background noise. BEM was fit by Weibull distribution if more than 5 non-zero non-reference alleles were observed at a tumor variant position across the reference samples. Otherwise, Gaussian distribution was used to fit BEM. Force-called variants were considered artifacts if the variant allele fraction (VAF) was not greater than BEM cutoff controlled by false discovery rate. Patients were considered MRD positive if the mean VAF was greater than the estimated limit of detection. The Kaplan-Meier (KM) estimator with log-rank test and Cox proportional-hazards model were used for survival analyses. Results: Background noise was observed in reference lymph samples across all 12 nucleotide substitution classes. Out of 299 single nucleotide variants (SNVs), 94 were determined as artifacts by BEM. Among the artifacts, 77 were C/G to A/T mutations, indicating that the majority of recurrent background artifact in TS is likely the result of oxidative damage. The artifacts had VAFs ranging from 0.004% to 0.12% with median of 0.02%. Incorporation of BEM in lymph TS showed significantly improved specificity (sensitivity (SN) = 71%, specificity (SP) = 65%; p = 0.027, Hazard ratio (HR) = 3.07) compared to the same cohort without BEM (SN = 76%, SP = 30%, p = 0.60, HR = 1.34). Conclusions: BEM is a sensitive and robust method to improve ctDNA detection accuracy in TS. We demonstrated its utility in improving recurrence prediction in a HNSCC cohort. Given its advantages, we envision that BEM will prove useful as a general strategy for deep sequencing applications requiring precise digital quantification of low frequency alleles in TS. Citation Format: Zhuosheng Gu, Maciej Pacula, Seka Lazare, Noah Earland, Marra S. Francis, Adam Harmon, Megan Long, Aadel A. Chaudhuri, Jose P. Zevallos, Wendy Winckler. Base-error model (BEM) for improved detection of ctDNA in lymph samples of HPV-negative head and neck cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B057.

Abstract A037: Improved liquid biopsy assay performance using novel highly accurate sequencing-by-binding (SBB) technology

Abstract Liquid biopsy is revolutionizing the field of early cancer detection research through non-invasive detection of tumor DNA in the blood. However, existing liquid biopsy assays are limited in their sensitivity for ctDNA detection at low variant allele frequencies (VAFs), with most relying on extreme sequencing depth and computational error correction to separate the true ctDNA signal from background errors. This limitation is particularly problematic in the area of early cancer detection and minimal residual disease monitoring, in which expected ctDNA allele frequencies are extremely low. Novel strategies are therefore needed to help improve liquid biopsy assay sensitivity and reduce per-sample sequencing requirements. Here we describe PacBio’s application of the Onso short-read sequencing system to enable detection of ctDNA at low VAFs using the SeraCare Complete ctDNA Mutation Mix reference standard. The Onso system makes use of a novel sequencing by binding (SBB) method to achieve 10- to 100-fold greater quality scores, with ≥90% of reads at Q40 or above. We performed targeted capture and sequencing of libraries prepared from the SeraCare reference mix diluted into WT human DNA at the following VAFs: 0.00% (WT), 0.05%, 0.10%, and 0.25%, and compared the sensitivity at each VAF for SBB compared to a competitor method using sequencing by synthesis (SBS) at varying sequencing depths. At equivalent sequencing depth (12,000X) we observed superior recall for low frequency variants (VAF = 0.05%, 0.1%) in the reference sample for Onso compared to SBS. Furthermore, SBB was able to achieve comparable sensitivity results to SBS using four- fold less sequencing (6,000X vs. 24,000X), and without the use of computational error correction. Combining SBB with error correction methods boosted sensitivity even further, suggesting an additive value for these technologies. Comparison of false positive variant calls in the WT samples showed a dramatic decrease in false positives for Onso (n=2) compared to SBS (n=49) across 10 subsampling replicates, and observed allele frequencies for Onso were closer to the expected values. In conclusion, Onso achieved superior sensitivity for ctDNA detection at compared to SBS at equivalent sequencing depths, and achieved similar performance with four- fold less sequencing and without the use of error correction. This improvement was due in part to the reduction in false positive variant calling in the WT sample, which lowers the limit of detection to maximize variant detection while maintaining high specificity. Taken together, our results demonstrate the potential of SBB to improve upon existing methods of liquid biopsy and better enable research on early cancer detection and minimal residual disease. Citation Format: Alexandra Sockell, Dan Nasko, Young Kim, Kristi Kim, Jonas Korlach. Improved liquid biopsy assay performance using novel highly accurate sequencing-by-binding (SBB) technology [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A037.

Abstract A057: Cancer-induced epigenomic changes in circulating immune cells enable breast, colorectal and lung cancer detection

Abstract Background: Epigenomic changes via the measurement of 5-hydroxymethylation cytosine (5hmC) in DNA have been shown to enable detection in a variety of cancers including pancreatic and ovarian cancer in plasma-derived cell-free DNA (cfDNA). Here, we investigated whether blood-based 5hmC profiling of genomic DNA (gDNA) derived from cells of the buffy coat (BC) possess signals that enable the detection of breast, colorectal and lung cancers. Method: Blood samples collected in Streck tubes from cancer patients or non-cancer controls were processed to obtain buffy coat and plasma samples. gDNA and cfDNA was isolated from buffy coat and plasma, respectively. gDNA or cfDNA was then subjected to 5hmC enrichment by click chemistry followed by preparation of sequencing libraries. Sequencing was performed to obtain genome-wide 5hmC profiles of buffy coat gDNA and cfDNA samples. Results: Differential hydroxymethylation analysis of BC gDNA revealed increased 5hmC over genes associated with myeloid lineage in breast, colorectal and lung cancers relative to non-cancer controls. Flow cytometry analysis confirmed increases in myeloid cells in cancer samples relative to non-cancer controls. 5hmC patterns in BC gDNA showed a larger number of cancer-associated changes compared to cfDNA, in particular this was found in early-stage disease. 5hmC features from BC gDNA or plasma-derived cfDNA were used to build prediction models that yielded cancer classification with overall sensitivity of 51.3% and 52.6% at 98% specificity, for BC or cfDNA, respectively. For early-stage cancer, the sensitivity at 98% specificity was 46.5% for the BC and 28.2% for cfDNA model. A final prediction model incorporating features from both tissue sources achieved superior classification (65.8% sensitivity) at 98% specificity, compared with using features from either tissue source alone, particularly for early-stage cancer which achieved 53.5% sensitivity of cancer detection. Conclusion: Our results demonstrate that cancer impacts the 5hmC profiles of buffy coat cells which can be leveraged for cancer detection using blood. Citation Format: Gulfem D Guler, Giuliana P Mognol, Yuhong Ning, Carolina Fraire, David Haan, Michael Kesling, Roger Malta, Melissa Peters, Anna Bergamaschi, Tierney Phillips, Shimul Chowdhury, Wayne Volkmuth, Samuel Levy. Cancer-induced epigenomic changes in circulating immune cells enable breast, colorectal and lung cancer detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A057.

Cross-comparison of gut metagenomic profiling strategies

The rapid advancements in sequencing technologies and bioinformatics have enabled metagenomic research of complex microbial systems, but reliable results depend on consistent laboratory and bioinformatics approaches. Current efforts to identify best practices often focus on optimizing specific steps, making it challenging to understand the influence of each stage on microbial population analysis and compare data across studies. This study evaluated DNA extraction, library construction methodologies, sequencing platforms, and computational approaches using a dog stool sample, two synthetic microbial community mixtures, and various sequencing data sources. Our work, the most comprehensive evaluation of metagenomic methods to date. We developed a software tool, termed minitax, which provides consistent results across the range of platforms and methodologies. Our findings showed that the Zymo Research Quick-DNA HMW MagBead Kit, Illumina DNA Prep library preparation method, and the minitax bioinformatics tool were the most effective for high-quality microbial diversity analysis. However, the effectiveness of pipelines or method combinations is sample-specific, making it difficult to identify a universally optimal approach. Therefore, employing multiple approaches is crucial for obtaining reliable outcomes in microbial systems.

A chromosome-level genome assembly of a model conifer plant, the Japanese cedar, Cryptomeria japonica D. Don.

The Japanese cedar (Cryptomeria japonica D. Don) is one of the most important Japanese forest trees, occupying approximately 44% of artificial forests and planted in East Asia, the Azores Archipelago, and certain islands in the Indian Ocean. Although the huge genome of the species (ca. 9 Gbp) with abundant repeat elements may have represented an obstacle for genetic analysis, this species is easily propagated by cutting, flowered by gibberellic acid, transformed by Agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica recommend it as a model conifer species for which reference genome sequences are necessary. Herein, we report the first chromosome-level assembly of C. japonica (2n = 22) using third-generation selfed progeny (estimated homozygosity rate = 0.96). Young leaf tissue was used to extract high molecular weight DNA (> 50kb) for HiFi PacBio long-read sequencing and to construct an Hi-C/Omni-C library for Illumina short-read sequencing. The 29× and 26× genome coverage of HiFi and Illumina reads, respectively, for de novo assembly yielded 2,651 contigs (9.1 Gbp, N50 contig size 12.0 Mbp). Hi-C analysis mapped 97% of the nucleotides on 11 chromosomes. The assembly was verified through comparison with a consensus linkage map comprising 7,781 markers. BUSCO analysis identified ∼ 91% conserved genes. Annotations of genes and comparisons of repeat elements with other Cupressaceae and Pinaceae species provide a fundamental resource for conifer research.

Seq-Scope: repurposing Illumina sequencing flow cells for high-resolution spatial transcriptomics.

Spatial transcriptomics technologies aim to advance gene expression studies by profiling the entire transcriptome with intact spatial information from a single histological slide. However, the application of spatial transcriptomics is limited by low resolution, limited transcript coverage, complex procedures, poor scalability and high costs of initial setup and/or individual experiments. Seq-Scope repurposes the Illumina sequencing platform for high-resolution, high-content spatial transcriptome analysis, overcoming these limitations. It offers submicrometer resolution, high capture efficiency, rapid turnaround time and precise annotation of histopathology at a much lower cost than commercial alternatives. This protocol details the implementation of Seq-Scope with an Illumina NovaSeq 6000 sequencing flow cell, allowing the profiling of multiple tissue sections in an area of 7 mm × 7 mm or larger. We describe the preparation of a fresh-frozen tissue section for both histological imaging and sequencing library preparation and provide a streamlined computational pipeline with comprehensive instructions to integrate histological and transcriptomic data for high-resolution spatial analysis. This includes the use of conventional software tools for single-cell and spatial analysis, as well as our recently developed segmentation-free method for analyzing spatial data at submicrometer resolution. Aside from array production and sequencing, which can be done in batches, tissue processing, library preparation and running the computational pipeline can be completed within 3 days by researchers with experience in molecular biology, histology and basic Unix skills. Given its adaptability across various biological tissues, Seq-Scope establishes itself as an invaluable tool for researchers in molecular biology and histology.

Amplicon-based long-read sequencing is useful for confirming zygosity of DSG2 variants associated with Japanese ARVC

Abstract Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy mainly caused by desmosomal gene variants. Previously, we reported that DSG2 variants were the most common in 153 Japanese ARVC probands (DSG2 multiple, n=62 and single, n=4; PKP2, n=28; others, n=7). Among the multiple DSG2 variant carriers, 22 probands were compound heterozygous carriers, and 25 were homozygous, which were confirmed by genetic testing for family members (Figure A). On the other hand, 88% of their family members with single DSG2 variant carriers were not diagnosed with ARVC, which meant that recessive DSG2 variants were the major cause of ARVC in Japan. However, zygosities in remaining 15 probands with multiple DSG2 variants were unknown due to the lack of genome data from their parents. Purpose This study aimed to clarify the zygosities of multiple DSG2 variants using amplicon-based long-read sequencing. Methods Genomic DNA was extracted from whole blood using standard protocols. The range containing multiple variants was amplified by long-range PCR. Sequencing libraries were prepared using ONT Ligation Sequencing Kit. Each library was loaded onto a flow cell for sequencing on ONT GridION Mk1 running MinKNOW control software. FASTQ files were aligned to hg38 using minimap2. Variants were called using Clair3 and phased using WhatsHap. Integrative Genomics Viewer (IGV) was used for visualization of the phased variants. Results We successfully phased all the multiple DSG2 variants found in the 15 proband. A typical result of amplicon-based LRS was shown in Figure B. It was IGV screen shot of reads at DSG2 containing c.874C>T (p.R292C) and c.1481A>C (p.D494A). The reads colored in red belonged to one haplotype, while the ones in blue belonged to the other. Each of the two variants was on different color reads, indicating they were compound heterozygous variants. Using LRS, we confirmed all the 15 probands had compound heterozygous variants in DSG2. Overall, the 62 probands (40.5%) with DSG2 variants were recessive variant carriers (Figure A). Conclusions Japanese ARVC was mainly caused by autosomal recessive variants in DSG2. Clarifying the zygosity of causative variants is crucial for the diagnosis in recessive diseases including Japanese ARVC. Amplicon-based LRS was helpful for phasing the multiple DSG2 variants directly without genetic testing of the parents.Figure AFigure B

RNA interference protocols for gene silencing in the spittlebug Philaenus spumarius, vector of Xylella fastidiosa

RNA interference (RNAi) is double stranded RNA (dsRNA)-based gene silencing mechanism. Exogenous dsRNAs application to crops has raised as a powerful tool to control agricultural pests. In particular, several sap-feeder are important plant pathogens vectors, such as Philaenus spumarius, known as main vector of Xylella fastidiosa (Xf), causal agent of olive quick decline syndrome (OQDS) in southern Italy. Here, dsATP synthase beta (dsATP), dsLaccase (dsLacc) and dsGreen Fluorescent Protein (dsGFP) as control, were provided to spittlebug adults by microinjection or to nymphs fed on dsRNA-treated plant shoots. Treated insects were collected at different time points to monitor silencing efficiency over time, describing significant reduction of transcript levels from 8 to 24 days post treatment. Downregulation of target genes ranged from 2- to 16-fold compared to the corresponding dsGFP controls, where highest silencing effects were generally noticed for ATP synthase beta. Sequencing of libraries obtained from total smallRNA (sRNA) showed the generation of dsRNA-derived sRNAs by RNAi pathway, with majority of reads mapping exclusively on the correspondent dsRNA. Also, we characterized components of a functional RNAi machinery in P. spumarius. Further research is needed to clarify such mechanism, screen effective target lethal genes to reduce vector population and improve delivery strategies.

High prevalence of cardiotropic viruses in endomyocardial biopsies from patients with inflammatory cardiomyopathy demonstrated by a novel targeted NGS approach

Abstract Background Viral infection of the heart muscle is a common cause of myocarditis and viral persistence can lead to chronic inflammatory cardiomyopathies (DCMi). A number of viruses including parvovirus B19 (B19V), adenovirus (AdV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes virus 6 (HHV-6) and enteroviruses (EV) are known to infect the myocardium, but their prevalence and role in persisting DCMi are not well understood. Virological etiologies can only be determined via endomyocardial biopsy (EMB), but the low tissue amount limits the number of conventional viral nucleic acid tests (virus-specific PCR) that can be performed in parallel. This study aimed to detect known and novel cardiotropic viruses in EMB of DCMi patients using a targeted next generation sequencing (NGS) approach. Methods A cohort of 33 patients with inflammatory cardiomyopathy (DCMi) (LVEF=29±12%) were retrospectively analyzed and compared to previous results of virus-specific PCR. DNA and RNA extracted from EMB were used for sequencing library preparation, and viral nucleic acids were enriched via a custom myBaits hybridization capture approach. Enriched libraries were sequenced on an Illumina MiSeq-platform in paired-end mode (500.000 reads/sample), and reads were taxonomically classified via Kraken2 and mapped against viral reference genomes using BWA-MEM2 or HiSat2, and subsequently deduplicated. Viral reads detected via both Kraken2 and mapping were classified as valid. Results Targeted NGS confirmed 19 out of 22 (86%) of the previous PCR-based viral detections. However, NGS was able to detect B19V DNA and RNA in an additional 11 and 15 patient samples respectively; CMV DNA/RNA in additional 5 and 4 samples respectively; EBV DNA/RNA in additional 5 and 1 samples respectively; and HHV-6 DNA/RNA in additional 3 and 1 samples respectively. Furthermore, viral reads of adenovirus type 2, adenovirus-associated virus type 2 (AAV-2), herpes viruses 7 and 8, and human parvovirus 4 were also found among the cohort. Thus, targeted NGS was able to identify 82 viral infections in a cohort of 33 DCMi patients, while only 22 were found using routine PCR tests. For routinely tested B19V, the use of the new NGS diagnostic approach improved sensitivity by 42% and specificity by 72%. Conclusions The presented NGS method represents a powerful new tool for the diagnosis of viral and inflammatory heart disease. The sensitivity is significantly higher than with conventional PCR approaches and the taxonomic classification is more specific. This enables the detection of previously false-negative routine viruses as well as potentially new cardiotropic pathogens from limited bioptic patient material – thus significantly increasing the diagnostic yield of EMB. In addition, our study demonstrates a high prevalence of non-enteroviral cardiotropic viruses in DCMi patients, shedding new light on the etiology of this heterogenous diseases and paving the way for more specific future treatments.

A single-stranded based library preparation method for virome characterization

BackgroundThe gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent diversity of genome types (dsDNA, ssDNA, dsRNA, and ssRNA) and topologies (linear, circular, or fragments), with subsequently biases associated with current sequencing library preparation methods. To overcome these problems and improve reproducibility and comparability across studies, universal or standardized virome sequencing library construction methods are highly needed in the gut virome study.ResultsWe repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA + Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches.ConclusionWe introduce and improve a fast, simple, and efficient ligation-based single-stranded DNA library preparation for gut virome study. This method is compatible with Illumina sequencing platforms and only requires ligation reagents within 3-h library preparation, which is similar or even better than the advanced library preparation method (xGen). We hope this method can be further optimized, validated, and widely used to make gut virome study more comparable and reproducible.EMStnH64sPQJC5VtA73CKfVideo

Assessment of heat tolerance and identification of miRNAs during high-temperature response in grapevine.

With global warming, heat stress has been recognized as a significant factor limiting grapevine development and fruit quality. MicroRNAs (miRNAs) are a class of small non-coding RNAs known to play crucial regulatory roles in stress resistance. Hence, there is an immediate requirement to cultivate and identify grapevine varieties that are resistant to heat and explore miRNA-mediated heat stress defense mechanisms. In this study, we assessed the thermal resistance of 38 grape germplasm resources and identified a series of miRNAs involved in heat stress resistance. The CK (25°C) and HS (45°C) groups of "Shenyue" cuttings of grapes were used as experimental materials for next-generation sequencing and construct libraries of small RNAs. A total of 177 known and 20 novel miRNAs were detected in the libraries. Differential expression analysis identified 65 differentially expressed miRNAs (DEMs) using the DE-Seq procedure. Furthermore, RT-qPCR validation confirmed complementary expression profiles of eight DEMs and their target genes between the HS and CK groups. Heterologous transformation further identified the function of Vvi-miR3633a downregulated under heat stress in Arabidopsis. In the heterologous expression lines, the survival rate was reduced by high temperature treatment indicating the ability of Vvi-miR3633a to regulate heat resistance. Assessing the heat resistance of grape species and the expression patterns of miRNA in response to high temperatures may reveal the molecular processes of heat resistance regulation mediated by miRNA in grapes under heat stress.

Template switching enables chemical probing of native RNA structures.

RNAs are often studied in non-native sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods - a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added post-probing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5' and 3' ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced non-native interactions in the full length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to non-native sequence elements.

Mouse Corneal Immune Cell Heterogeneity Revealed by Single-Cell RNA Sequencing.

This study aimed to define the heterogeneity, spatial localization, and functional roles of immune cells in the mouse cornea using single-cell RNA sequencing (scRNA-seq) and immunofluorescent staining. Enriched mouse corneal immune cells (C57BL/6 strain, age 16-20 weeks) underwent single-cell RNA sequencing library preparation, sequencing, and analysis with Seurat, Monocle 3, and CellChat packages in R. Pathway analysis used Qiagen Ingenuity Pathway Analysis software. Immunostaining confirmed cell distribution. We identified 14 distinct immune cell clusters (56% myeloid and 44% lymphoid). Myeloid populations included resident macrophages, conventional dendritic cells (cDC2s), Langerhans cells, neutrophils, monocytes, and mast cells. Additionally, lymphocyte subsets (B, CD8, CD4, γδT, natural killer, natural killer T, and group 2 innate lymphoid cells) were found. We also found three new subtypes of resident macrophages in the cornea. Trajectory analysis suggested a differentiation pathway from monocytes to conventional dendritic cells, resident macrophages, and LCs. Intercellular communication network analysis using cord diagram identified amyloid precursor protein, chemokine (C-C motif) ligands (2, 3, 4, 6, 7, 9, and 12), Cxcl2, Mif, Tnf, Tgfb1, Igf1, and Il10 as prominent ligands involved in these interactions. Sexually dimorphic gene expression patterns were observed, with male myeloid cells expressing genes linked to immune regulation (Egr1, Foxp1, Mrc1, and Il1rn) and females showing higher expression of antigen presentation genes (Clic1, Psmb8, and Psmb9). Finally, immunostaining confirmed the spatial distribution of these cell populations within the cornea. This study unveils a diverse immune landscape in the mouse cornea, with evidence for cell differentiation and sex-based differences. Immunostaining validates the spatial distribution of these populations, furthering our knowledge of corneal immune function.

Construction of competitive endogenous RNA network and identification of potential regulatory axis in vascular calcification.

Competitive endogenous RNAs (ceRNA) theory has been proved in numerous biological processes. Nevertheless, there is a lack of research applying the ceRNA theory to the study of vascular calcification (VC) in chronic kidney diseases (CKD). In the present study, a ceRNA network was constructed after conducting transcriptome sequencing of differentially expressed genes, followed by experimental validation to identify a new target for the diagnosis and treatment of vascular calcification. Total RNA was extracted from β-glycerophosphate (β-GP) cultured vascular smooth muscle cells (VSMCs) on Day 7. Illumina HiSeq platform was utilized to build sequencing libraries. GO and KEGG analysis was conducted to identify the function of the differentially expressed genes. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. A ceRNA network was established based on TargetScan, miRDB, miRWALK, and miRanda database. Western blot and qRT-PCR were used to explore the expression level of protein and RNA, respectively. The direct binding sites were confirmed by dual-luciferase reporter assay. In total, 647 differentially expressed lncRNAs and 289 differentially expressed mRNAs were identified (|log2FC| ≥ 1, p < .05). The function of differentially expressed mRNAs was mainly enriched in negative regulation of osteoblast differentiation, regulation of RNA metabolic process, and other typical pathways. The ceRNA network was generated with a total of 107 interaction pairs. The lncRNA Prrc2c/miR-145-5p/Smad3 axis was considered a potential regulatory pathway within the ceRNA network. The regulatory relationship and targets of this ceRNA axis were validated via invitro experiments. For the first time, we found that lncRNA Prrc2c was highly expressed and promoted calcification of VSMCs. Luciferase reporter assay showed that lncRNA Prrc2c could bind miR-145-5p at site 1755-1761. Similarly, luciferase reporter assay showed that miR-145-5p inhibited Smad3 expression by binding to its 3'UTR. Our findings provide a comprehensive examination of the ceRNA networks in vascular smooth muscle cells (VSMCs) treated with high phosphorate. Specifically, we have identified the role of lncRNA Prrc2c in promoting VSMC calcification through the miR-145-5p/Smad3 axis.

Genotype from Phenotype: Using CRISPR Screens to Dissect Lymphoma Biology.

Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, select cells with a specified phenotype, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human B-cell lymphoma cell lines but are highly amenable for other experimental purposes.

Systematic benchmark of single-cell hashtag demultiplexing approaches reveals robust performance of a clustering-based method.

Single-cell technology opened up a new avenue to delineate cellular status at a single-cell resolution and has become an essential tool for studying human diseases. Multiplexing allows cost-effective experiments by combining multiple samples and effectively mitigates batch effects. It starts by giving each sample a unique tag and then pooling them together for library preparation and sequencing. After sequencing, sample demultiplexing is performed based on tag detection, where cells belonging to one sample are expected to have a higher amount of the corresponding tag than cells from other samples. However, in reality, demultiplexing is not straightforward due to the noise and contamination from various sources. Successful demultiplexing depends on the efficient removal of such contamination. Here, we perform a systematic benchmark combining different normalization methods and demultiplexing approaches using real-world data and simulated datasets. We show that accounting for sequencing depth variability increases the separability between tagged and untagged cells, and the clustering-based approach outperforms existing tools. The clustering-based workflow is available as an R package from https://github.com/hwlim/hashDemux.

RpoS activates formation of Salmonella Typhi biofilms and drives persistence in the gall bladder.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway established in S. Typhimurium. We undertook a genome-wide Tn5 mutation screen in H58, a clinically relevant multidrug resistance strain of S. Typhi, in gallstone-mimicking conditions. We generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. An rpoS null mutant failed to colonize the gall bladder in chronic zebrafish infections. Overall, our work uncovered a novel RpoS-driven, CsgD-independent paradigm for the formation of cholesterol-attached Typhi biofilms, and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections. Our identification of the biofilm regulators in S. Typhi paves the way for the development of drugs against typhoid carriage, which will ultimately control the increased incidence of gall bladder cancer in typhoid carriers.

Integrating targeted genetic markers to genotyping-by-sequencing for an ultimate genotyping tool.

New selection methods, using trait-specific markers (marker-assisted selection (MAS)) and/or genome-wide markers (genomic selection (GS)), are becoming increasingly widespread in breeding programs. This new era requires innovative and cost-efficient solutions for genotyping. Reduction in sequencing cost has enhanced the use of high-throughput low-cost genotyping methods such as genotyping-by-sequencing (GBS) for genome-wide single-nucleotide polymorphism (SNP) profiling in large breeding populations. However, the major weakness of GBS methodologies is their inability to genotype targeted markers. Conversely, targeted methods, such as amplicon sequencing (AmpSeq), often face cost constraints, hindering genome-wide genotyping across a large cohort. Although GBS and AmpSeq data can be generated from the same sample, an efficient method to achieve this is lacking. In this study, we present the Genome-wide & Targeted Amplicon (GTA) genotyping platform, an innovative way to integrate multiplex targeted amplicons into the GBS library preparation to provide an all-in-one cost-effective genotyping solution to breeders and research communities. Custom primers were designed to target 23 and 36 high-value markers associated with key agronomical traits in soybean and barley, respectively. The resulting multiplex amplicons were compatible with the GBS library preparation enabling both GBS and targeted genotyping data to be produced efficiently and cost-effectively. To facilitate data analysis, we have introduced Fast-GBS.v3, a user-friendly bioinformatic pipeline that generates comprehensive outputs from data obtained following sequencing of GTA libraries. This high-throughput low-cost approach will greatly facilitate the application of DNA markers as it provides required markers for both MAS and GS in a single assay.

Multiplexed chromatin immunoprecipitation sequencing for quantitative study of histone modifications and chromatin factors.

ChIP-seq is a widely used technique for studying histone post-translational modifications and DNA-binding proteins. DNA fragments associated with a specific protein or histone modification epitope are captured by using antibodies, sequenced and mapped to a reference genome. Albeit versatile and popular, performing many parallel ChIP-seq experiments to compare different conditions, replicates and epitopes is laborious, is prone to experimental variation and does not allow quantitative comparisons unless adequate spike-in chromatin is included. We present a detailed protocol for performing and analyzing a multiplexed quantitative chromatin immunoprecipitation-sequencing experiment (MINUTE-ChIP), in which multiple samples are profiled against multiple epitopes in a single workflow. Multiplexing not only dramatically increases the throughput of ChIP-seq experiments (e.g., profiling 12 samples against multiple histone modifications or DNA-binding proteins in a single experiment), but also enables accurate quantitative comparisons. The protocol consists of four parts: sample preparation (i.e., lysis, chromatin fragmentation and barcoding of native or formaldehyde-fixed material), pooling and splitting of the barcoded chromatin into parallel immunoprecipitation reactions, preparation of next-generation sequencing libraries from input and immunoprecipitated DNA and data analysis using our dedicated analysis pipeline. This pipeline autonomously generates quantitatively scaled ChIP-seq tracks for downstream analysis and visualization, alongside necessary quality control indicators. The entire workflow requires basic knowledge in molecular biology and bioinformatics and can be completed in 1 week. MINUTE-ChIP empowers biologists to perform every ChIP-seq experiment with an appropriate number of replicates and control conditions, delivering more statistically robust, exquisitely quantitative and biologically meaningful results.