Sequencing Library Preparation Research Articles

Basics of high throughput sequencing Summary

Sequencing can provide genomic characterisation of a specific organism, as well as of a whole environmental or clinical sample. High Throughput Sequencing (HTS) makes it possible to generate an enormous amount of genomic data at gradually decreasing costs and almost in real-time. HTS is used, among others, in medicine, veterinary medicine, microbiology, virology and epidemiology. The paper presents practical aspects of the HTS technology. It describes generations of sequencing, which vary in throughput, read length, accuracy and costs ̶ and thus are used for different applications. The stages of HTS, as well as their purposes and pitfalls, are presented: extraction of the genetic material, library preparation, sequencing and data processing. For success of the whole process, all stages need to follow strict quality control measurements. Choosing the right sequencing platform, proper sample and library preparation procedures, as well as adequate bioinformatic tools are crucial for high quality results.

Critical Steps in Shotgun Metagenomics-Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing.

Shotgun metagenomics offers a broad detection of pathogens for rapid blood stream infection of pathogens but struggles with often low numbers of pathogens combined with high levels of human background DNA in clinical samples. This study aimed to develop a shotgun metagenomics protocol using blood spiked with various bacteria and to assess bacterial DNA extraction efficiency with human DNA depletion. The Blood Pathogen Kit (Molzym) was used to extract DNA from EDTA-whole blood (WB) and plasma samples, using contrived blood specimens spiked with bacteria for shotgun metagenomics diagnostics via Oxford Nanopore sequencing and PCR-based library preparation. Results showed that bacterial reads were higher in WB than plasma. Differences for Staphylococcus aureus and Streptococcus pneumoniae were more pronounced compared to Escherichia coli. Plasma samples exhibited better method reproducibility, with more consistent droplet digital PCR results for human DNA. The study found that extraction was more efficient for Gram-positive bacteria than Gram-negative, suggesting that the human DNA depletion exerts a negative effect on Gram-negative bacteria. Overall, shotgun metagenomics needs further optimisation to improve bacterial DNA recovery and enhance pathogen detection sensitivity. This study highlights some critical steps in the methodology of shotgun metagenomic-based diagnosis of blood stream infections using Nanopore sequencing.

Multiplexing cortical brain organoids for the longitudinal dissection of developmental traits at single-cell resolution.

Dissecting human neurobiology at high resolution and with mechanistic precision requires a major leap in scalability, given the need for experimental designs that include multiple individuals and, prospectively, population cohorts. To lay the foundation for this, we have developed and benchmarked complementary strategies to multiplex brain organoids by pooling cells from different pluripotent stem cell (PSC) lines either during organoid generation (mosaic models) or before single-cell RNA sequencing (scRNA-seq) library preparation (downstream multiplexing). We have also developed a new computational method, SCanSNP, and a consensus call to deconvolve cell identities, overcoming current criticalities in doublets and low-quality cell identification. We validated both multiplexing methods for charting neurodevelopmental trajectories at high resolution, thus linking specific individuals' trajectories to genetic variation. Finally, we modeled their scalability across different multiplexing combinations and showed that mosaic organoids represent an enabling method for high-throughput settings. Together, this multiplexing suite of experimental and computational methods provides a highly scalable resource for brain disease and neurodiversity modeling.

An Improved RNA Extraction Protocol for Rye Grain Full-Length Transcriptome Sequencing.

RNA quality and integrity are critical for many studies in plant molecular biology. However, extracting high-quality RNA from cereal grains is challenging due to the presence of polysaccharides, polyphenols, and other compounds that bind or coprecipitate with RNA particles. To address this, we introduced an initial purification step into the Tri Reagent Solution protocol, which effectively eliminated starch and other contaminants. The performance of this modified protocol was then compared with five other RNA extraction methods, including those based on Tri Reagent Solution and the Direct-zol RNA Miniprep Plus Kit. Introducing our method modification prior to homogenization with Tri Reagent Solution successfully yielded total RNA with both high quality (RIN values ranging from 9.50 to 9.70) and high efficiency, making it suitable for both mRNA extraction using the Dynabeads mRNA Purification Kit and library preparation for transcriptome sequencing by long-read methods, such as Oxford Nanopore Technologies. The protocol was successfully applied to total RNA extraction from rye grains at 14 and 21 days after pollination. This study demonstrates that improving the Tri Reagent Solution protocol through initial purification enables the extraction of high-quality RNA from rye grains.

Improved precision, sensitivity, and adaptability of ordered two-template relay cDNA library preparation for RNA sequencing.

Sequencing RNAs that are biologically processed or degraded to less than ∼100 nt typically involves multistep, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced ordered two-template relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced nonproductive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 pg (<1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or posttranscriptional modifications.

MuMu: a sample multiplexing protocol for droplet-based simultaneous single nuclei RNA- and ATAC-seq systems.

SummarySample multiplexing is a common approach to reduce experimental cost and technical batch effect. Here, we present a protocol that for the first time allows the pooling of single nuclei from multiple biological samples prior to performing simultaneous single nuclei RNA-seq and ATAC-seq, which we termMultiplexedMultiome (MuMu). We describe steps for assembling the custom Tn5 transposome, performing the transposition reaction, nuclei pooling, sequencing library preparation, and sequencing data pre-processing. This protocol will greatly reduce the cost of sn-Multiome.Graphical abstract

Genomic surveillance of multidrug-resistant organisms based on long-read sequencing

BackgroundMultidrug-resistant organisms (MDRO) pose a significant threat to public health worldwide. The ability to identify antimicrobial resistance determinants, to assess changes in molecular types, and to detect transmission are essential for surveillance and infection prevention of MDRO. Molecular characterization based on long-read sequencing has emerged as a promising alternative to short-read sequencing. The aim of this study was to characterize MDRO for surveillance and transmission studies based on long-read sequencing only.MethodsGenomic DNA of 356 MDRO was automatically extracted using the Maxwell-RSC48. The MDRO included 106 Klebsiella pneumoniae isolates, 85 Escherichia coli, 15 Enterobacter cloacae complex, 10 Citrobacter freundii, 34 Pseudomonas aeruginosa, 16 Acinetobacter baumannii, and 69 methicillin-resistant Staphylococcus aureus (MRSA), of which 24 were from an outbreak. MDRO were sequenced using both short-read (Illumina NextSeq 550) and long-read (Nanopore Rapid Barcoding Kit-24-V14, R10.4.1) whole-genome sequencing (WGS). Basecalling was performed for two distinct models using Dorado-0.3.2 duplex mode. Long-read data was assembled using Flye, Canu, Miniasm, Unicycler, Necat, Raven, and Redbean assemblers. Long-read WGS data with > 40 × coverage was used for multi-locus sequence typing (MLST), whole-genome MLST (wgMLST), whole-genome single-nucleotide polymorphisms (wgSNP), in silico multiple locus variable-number of tandem repeat analysis (iMLVA) for MRSA, and identification of resistance genes (ABRicate).ResultsComparison of wgMLST profiles based on long-read and short-read WGS data revealed > 95% of wgMLST profiles within the species-specific cluster cut-off, except for P. aeruginosa. The wgMLST profiles obtained by long-read and short-read WGS differed only one to nine wgMLST alleles or SNPs for K. pneumoniae, E. coli, E. cloacae complex, C. freundii, A. baumannii complex, and MRSA. For P. aeruginosa, differences were up to 27 wgMLST alleles between long-read and short-read wgMLST and 0–10 SNPs. MLST sequence types and iMLVA types were concordant between long-read and short-read WGS data and conventional MLVA typing. Antimicrobial resistance genes were detected in long-read sequencing data with high sensitivity/specificity (92–100%/99–100%). Long-read sequencing enabled analysis of an MRSA outbreak.ConclusionsWe demonstrate that molecular characterization of automatically extracted DNA followed by long-read sequencing is as accurate compared to short-read sequencing and suitable for typing and outbreak analysis as part of genomic surveillance of MDRO. However, the analysis of P. aeruginosa requires further improvement which may be obtained by other basecalling algorithms. The low implementation costs and rapid library preparation for long-read sequencing of MDRO extends its applicability to resource-constrained settings and low-income countries worldwide.

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.

BIOM-04. SINGLE-CELL RNA SEQUENCING PROTOCOL OF THE HUMAN METASTATIC PROSTATE SPINE TUMOR MICROENVIRONMENT

Abstract INTRODUCTION 90% of spine tumor patients can develop metastases with debilitating complications, such as sensorimotor dysfunction, paralysis, and spinal instability. As spine tumor patients have limited eligibility to participate in clinical trials and have heterogenous tumor pathology, there is a distinct lack of therapeutic targets and prognostication markers of disease. Thus, there is a distinct need for molecular profiling of spine tumors. OBJECTIVE We describe an OR-to-bench-top processing of spine tumors for single-cell omic studies, with samples from a metastatic prostate cancer patient to demonstrate the ability to compare molecular markers and microenvironments of difficult-to-process spine tumors and adjacent normal tissue. METHODS Under IRB protocol #Pro00101198, patients undergoing spine surgery with the Department of Neurosurgery at Duke University were consented for tissue collection. Vertebral osseous metastases and adjacent normal tissue were confirmed histologically (H&amp;E, immunohistochemistry) and radiologically (MRI, PET, CT, and x-ray). Tissues were manually and enzymatically homogenized in serum-free media with collagenase A and DNAse I to obtain a single cell suspension. Cells were then filter sterilized, erythrolyzed, counted to determine live-death ratio, resuspended to 10-20 million cells per mL, then cryopreserved for downstream standard library preparation for single-cell sequencing using the 10X genomics platform. Downstream analysis was performed using Seurat pipeline, InferCNV analysis, and SingleR. RESULTS P404S and PSA expression confirmed tumor was prostate in origin. Radiological imaging and H&amp;E staining confirmed the presence of T7 osseous metastasis. Uniform manifold approximation and project (UMAP) plots identified 12 unique clusters of immune cell subpopulations between normal and tumorous vertebrae, with their own subsets of differentially expressed genes. CONCLUSION We have developed a protocol to generate granular single cell molecular profiling for spine tumors and metastases. Applications of this protocol can aid in identifying therapeutic targets, molecular predictors of disease, and potentiate increased eligibility of patients for clinical trials.

Improved precision, sensitivity, and adaptability of Ordered Two-Template Relay cDNA library preparation for RNA sequencing.

Sequencing RNAs that are biologically processed or degraded to less than ~100 nucleotides typically involves multi-step, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced Ordered Two-Template Relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced non-productive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 picograms (less than 1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or post-transcriptional modifications.

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.

Genomic Characterization of SARS-CoV-2 Isolates Obtained from Antalya, Türkiye

As the number of coronavirus diseases-2019 (COVID-19) cases have decreased and measures have started to be implemented at an individual level rather than in the form of social restrictions, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) still maintains its importance and has already taken its place in the spectrum of agents investigated in multiplex molecular test panels for respiratory tract infections in routine diagnostic use. In this study, we aimed to present mutation analysis and clade distribution of whole genome sequences from randomly selected samples that tested positive with SARS-CoV-2 specific real-time reverse transcription polymerase chain reaction (rRT-PCR) test at different periods of the pandemic in our laboratory with a commercial easy-to-use kit designed for next-generation sequencing systems. A total of 84 nasopharyngeal/oropharyngeal swab samples of COVID-19 suspected patients which were sent for routine diagnosis to the medical microbiology laboratory and detected as SARSCoV-2 RNA positive with rRT-PCR were randomly selected from different periods for sequence analysis. Library preparation for sequencing was performed with the commerical EasySeq SARS-CoV-2 RC PCR kit (Nimagen, the Netherlands). The data generated from the Illumina MiSeq system (Illumina Inc, San Diego, CA, USA) were analysed using CLC Genomics Workbench (CLC, Qiagen, Hilden, Germany). Nextstrain clades detected in order of frequency were 21J (Delta) (25%, n= 21), 21L (Omicron) (23.8%, n= 20), 20B (19%, n= 16), 20A (15.5%, n= 13), 21K (Omicron) (11.9%, n= 10), 19A (3.6%, n= 3), and 22B (Omicron) (1.2%, n= 1). Excluding one patient sample which was identified as 22B (Omicron), a total of 2829 common distinct mutations (2076 missense, 551 synonymous, 192 deletions and 10 insertions) were detected. 100 mutations were observed in the non-coding 5' untranslated region (UTR). The majority of the mutations were located in the Spike gene region (1120 mutations), followed by the ORF1a (624 mutations), nucleocapside (315 mutations) and ORF1b (263 mutations) gene regions. Sampling times of the patients were March 2020 (n= 1), April 2020 (n= 11), May 2020 (n= 1), June 2020 (n= 2), July 2020 (n= 3), August 2020 (n= 1), September 2020 (n= 5), November 2020 (n= 2), December 2020 (n= 6), December 2021 (n= 19), January 2022 (n= 11), March 2022 (n= 16), April 2022 (n= 3), and June 2022 (n= 3). As a result, in this study, SARS-CoV-2 variants and mutations in the Mediterranean Region of Türkiye, Antalya province were analyzed in detail. To the best of our knowledge, no SARS-CoV-2 genome analysis study from the pandemic period has been reported in our province. When the sequences from our study were uploaded to the GISAID Instant Audacity system and the related genomes obtained from different countries in the EpiCoV database metadata were examined, the top two countries in terms of similarity and which could be associated with the main entry route of the virus into Türkiye were Germany and the United Kingdom. In today's world, where it is discussed what can be done to be prepared for possible new pandemics based on the COVID-19 pandemic, the importance of being proactive in molecular surveillance studies is indisputable. Developing countries should be supported and encouraged to research new variants and share data in addition to known variants in pandemic control. At this point, we believe that past pandemic data reported from different geographical regions will also be valuable in terms of predicting the circulation network and taking precautions in a possible new pandemic.

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

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.

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.

Generation of Barcode-Tagged Vibrio fischeri Deletion Strains and Barcode Sequencing (BarSeq) for Multiplex Strain Competitions.

Vibrio fischeri is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package. Aspects of this protocol could be readily adapted for tagging wild-type V. fischeri strains with a neutral barcode for examination of population dynamics or BarSeq analyses in other species. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of the erm-bar DNA Basic Protocol 2: Generation of a targeted and barcoded deletion strain of V. fischeri Alternate Protocol: Parallel generation of multiple barcode-tagged V. fischeri deletion strains Basic Protocol 3: Setting up mixed populations of barcode-tagged strains Basic Protocol 4: Performing a competitive growth assay Basic Protocol 5: Amplicon library preparation and equimolar pooling Basic Protocol 6: Sequencing on Illumina iSeq 100 Basic Protocol 7: BarSeq data analysis.

Profiles of differential expression of miRNAs in the late stage of red blood cell preservation and their potential roles

ObjectiveTo detect the differentially expressed regulatory miRNAs in the late stage of red blood cell (RBC) preservation and predict their roles. MethodsSuspended RBCs with different storage periods of 35 day, 42 day, and 50 day were collected for routine blood tests, RNA extraction, and preparation of small RNA sequencing libraries. The constructed libraries were sequenced and the biological functions of differential miRNAs in RBCs in the late storage were analyzed by bioinformatics. ResultsRoutine indicators of RBCs in the late stage were not significantly affected by preservation time. The Pearson correlation analysis performing on RBC miRNAs with different storage days revealed that RBC miRNAs changed with the increase of storage days. RBC miRNAs from day 35 (D35), day 42 (D42) and day 50 (D50) showed significant differences (P < 0.05). Compared RBC miRNAs from D42 with these from D35, there were 690 up-regulated miRNAs and 82 down-regulated miRNAs; compared RBC miRNAs from D50 with these from D35, there were 638 up-regulated miRNAs and 123 down-regulated miRNAs; compared RBC miRNAs from D42 with these from D50, there were 271 up-regulated miRNAs and 515 down-regulated miRNAs. GO enrichment analysis of target genes of differential miRNAs were mainly involved in cell metabolism, biosynthesis, protein modification, gene expression and transcriptional regulation of biological processes. KEGG pathway enrichment analysis of miRNA target genes showed that differential miRNA target genes were closely related to pathways in cancer. ConclusionMiRNAs were differentially expressed in the late stage of RBC preservation, and may be involved in various biological processes, especially cancer.

High-quality genome assemblies for nine non-model North American insect species representing six orders (Insecta: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Neuroptera).

Field-collected specimens were used to obtain nine high-quality genome assemblies from a total of 10 insect species native to prairies and savannas of central Illinois (USA): Mellilla xanthometata (Lepidoptera: Geometridae), Stenolophus ochropezus (Coleoptera: Carabidae), Forcipata loca (Hemiptera: Cicadellidae), Coelinius sp. (Hymenoptera: Braconidae), Thaumatomyia glabra (Diptera: Chloropidae), Brachynemurus abdominalus (Neuroptera: Myrmeleontidae), Catonia carolina (Hemiptera: Achilidae), Oncometopia orbona (Hemiptera: Cicadellidae), Flexamia atlantica (Hemiptera: Cicadellidae) and Stictocephala bisonia (Hemiptera: Membracidae). Sequencing library preparation from single specimens was successful despite extremely small DNA yields (<0.1 μg) for some samples. Additional sequencing and assembly workflows were adapted to each sample depending on the initial DNA yield. PacBio circular consensus (CCS/HiFi) or continuous long reads (CLR) libraries were used to sequence DNA fragments up to 50 kb in length, with Illumina sequenced linked-reads (TellSeq libraries) and Omni-C libraries used for scaffolding and gap-filling. Assembled genome sizes ranged from 135 MB to 3.2 GB. The number of assembled scaffolds ranged from 47 to >13,000, with the longest scaffold per assembly ranging from ~23 to 439 Mb. Genome completeness was high, with BUSCO scores ranging from 85.5% completeness for the largest genome (Stictocephala bisonia) to 98.8% completeness for the smallest genome (Coelinius sp.). The unique content was estimated using RepeatMasker and GenomeScope2, which ranged from 50.7% to 75.8% and roughly decreased with increasing genome size. Structural annotation predicted a range of 19,281-72,469 protein models for sequenced species. Sequencing costs per genome at the time ranged from US$3-5k, averaged ~1600 CPU-hours on a high-performance cluster and required approximately 14 h of bioinformatics analyses with samples using PacBio HiFi data. Most assemblies would benefit from further manual curation to correct possible scaffold misjoins and translocations suggested by off-diagonal or depleted signals in Omni-C contact maps.

Targeted DNA-seq and RNA-seq of Reference Samples with Short-read and Long-read Sequencing

Next-generation sequencing (NGS) has revolutionized genomic research by enabling high-throughput, cost-effective genome and transcriptome sequencing accelerating personalized medicine for complex diseases, including cancer. Whole genome/transcriptome sequencing (WGS/WTS) provides comprehensive insights, while targeted sequencing is more cost-effective and sensitive. In comparison to short-read sequencing, which still dominates the field due to high speed and cost-effectiveness, long-read sequencing can overcome alignment limitations and better discriminate similar sequences from alternative transcripts or repetitive regions. Hybrid sequencing combines the best strengths of different technologies for a more comprehensive view of genomic/transcriptomic variations. Understanding each technology’s strengths and limitations is critical for translating cutting-edge technologies into clinical applications. In this study, we sequenced DNA and RNA libraries of reference samples using various targeted DNA and RNA panels and the whole transcriptome on both short-read and long-read platforms. This study design enables a comprehensive analysis of sequencing technologies, targeting protocols, and library preparation methods. Our expanded profiling landscape establishes a reference point for assessing current sequencing technologies, facilitating informed decision-making in genomic research and precision medicine.