Double-stranded cDNA Research Articles

P-2250. Metagenomic Next Generation Sequencing Enhances Rickettsial Infection Diagnosis in Patients with Acute Undifferentiated Fever

Abstract Background Murine and scrub typhus are common causes of acute undifferentiated fever (AUF) in tropical regions, after malaria, dengue fever, and typhoid fever have been ruled out. While indirect immunofluorescence assay (IFA) can diagnose both diseases, accuracy depends on when the sample is collected. To improve detection, we used unbiased metagenomic Next Generation Sequencing (mNGS) on 969 AUF patient samples with negative IFA results from Siriraj Hospital, Bangkok, Thailand collected between 2015 and 2021. Methods Plasma was pre-treated with benzonase before extraction on an Abbott m2000 instrument. mNGS libraries were prepared from double-stranded cDNA with Illumina Nextera XT reagents then sequenced on a NextSeq. Reads were taxonomically classified by Abbott pipelines SURPI or DiVir and mapped in CLC Bio Genomics Workbench (Qiagen, Inc). Results During the study period, a total of 2,465 samples were analyzed by IFA, identifying 58 (2.4%) cases of murine typhus and 26 (1.2%) cases of scrub typhus. Among IFA negative samples, mNGS identified Rickettsia spp. in 27 cases (2.8%), including R. typhi (murine typhus) (n=19), R. felis (n=5), and R. rickettsii, R. prowazaki, and R. australis (n=1 each). Notably, mNGS identified Orientia tsutsugamushi (scrub typhus) in one sample missed by IFA. For patients with mNGS-detected R. typhi, serological confirmation was achieved in 2 cases. Another 13 were classified as probable based on clinical presentation, while 4 cases lacked conclusive clinical correlation. For patients with R. felis identified by mNGS, 4/5 had confirmed or probable diagnoses based on clinical symptoms. The inconclusive case was a 65-year-old man with a urinary tract infection who died from sepsis for which mNGS revealed a co-infection with R. felis and Serratia marcescens. Interestingly, none of the mNGS positives for R. rickettsii, R. australis, and R. prowazaki had clinical presentations that supported the diagnoses. Conclusion mNGS significantly enhances the diagnosis of rickettsial infections, particularly for murine typhus. This improved diagnostic capability provided by mNGS holds promise for improved management of AUF patients, potentially reducing unnecessary testing and inappropriate antibiotic use. Disclosures Sonja Weiss, BS, Abbott Laboratories: Employee Julie Yamaguchi, BS, Abbott Laboratories: Employee Michael G. Berg, PhD, Abbott Laboratories: employee|Abbott Laboratories: Stocks/Bonds (Public Company) Francisco Averhoff, MD, MPH, Abbott Laboratories: Employee Gavin Cloherty, PhD, Abbott Laboratories: Employee

A comparative template-switching cDNA approach for HTS-based multiplex detection of three viruses and one viroid commonly found in apple trees

Exclusion is a keystone of integrated pest management to prevent the introduction of pathogens. U.S. plant quarantine programs employ PCR and high-throughput sequencing (HTS) to test imported plants for viruses and viroids of concern. Achieving a low limit of detection in any HTS protocol could be challenging. Following a template-switching cDNA amplification protocol, seven cDNA synthesis treatments were used to test simultaneously the relative abundance and coverage of the three most commonly latent RNA viruses found in apples: apple chlorotic leaf spot virus, apple stem grooving virus, and apple stem pitting virus, as well as the viroid apple hammerhead viroid. Amplified double-stranded cDNAs were subjected to library preparation using Nanopore SQK-DCS109 and Illumina Nextera XT, and sequenced with MinION and NextSeq2000, respectively. Treatments with oligo d(T)23-VN or its combination with random hexamers yielded the highest relative reads for viruses, while treatments containing the reverse primer pool produced more relative reads for AHVd. These treatments and random hexamers also generated the highest genome coverages, which were typically similar in both HTS workflows. However, relative abundances of viruses determined with SQK-DCS109 were up to 2.22-fold higher compared to Nextera XT. In contrast, Nextera XT yielded viroid reads 3.30-fold higher than SQK-DCS109. A framework of considerations for expanding this sensitive approach to other targets and crops is discussed.

Identification of Antisense RNA NRAS-AS and Its Preliminary Exploration of the Anticancer Regulatory Mechanism.

To explore the influence of NRAS-AS on the proliferation, apoptosis, cell cycle, migration, and invasion ability of HCC cells, as well as its underlying mechanisms. A double-stranded cDNA library for liver cancer cells was constructed, and identified NRAS-AS through High-throughput sequencing, bioinformatics, chain-specific fluorescent quantitative PCR, and RACE. NRAS-AS's effects on HepG2 and SMMC-7721 cells and gene expression were evaluated. Additionally, the study analyzed the influence of NRAS-AS overexpression on tumor formation in nude mice. Immunohistochemistry and Western blotting were used to detect NRAS protein levels in clinical samples. RT-qPCR examined NRAS-AS and NRAS gene expression in HCC and adjacent tissues. NRAS-AS overexpression suppresses HCC cell proliferation and invasion, induces cell cycle alterations in HepG2 and SMMC-7721 cells, and enhances apoptosis. NRAS-AS interference promoted liver cancer invasion, inhibited apoptosis, and influences the cell cycle. Nude mice overexpressing NRAS-AS showed smaller tumors. NRAS-AS expression in liver cancer patients correlated with clinical factors. RT-qPCR revealed an inverse correlation between NRAS-AS and NRAS gene expression in liver cancer and adjacent tissues. IHC analysis revealed reduced NRAS protein expression in HepG2 and SMMC-7721 cells following NRAS-AS overexpression. The impact of AZA treatment on antisense NRAS-AS and sense NRAS gene expression in liver cancer cells was observed, and antisense. Reduced NRAS-AS expression is frequently observed in HCC and is inversely related to NRAS gene expression, suggesting a role in HCC pathogenesis through NRAS regulation. Targeting antisense RNA NRAS-AS could hold promise as a therapeutic target and diagnostic biomarker for HCC.

X Ray-Induced Insulinoma Cell Line Rin-5F Has a Novel Mutation Site, C.A1459G (P.T487A), in Death Domain Associated Protein (DAXX) Gene

A popular toxicological and pharmacological research cell line is the insulin-secreting pancreatic cell line Rin-5F. The cell line originates from insulinomas induced by X-ray exposure. The author of this report looked at the mutation status of the DAXX gene in the Rin-5F cell line clone.<em> </em>The complete DNA and RNA were extracted from the cultivated cells as well. Double-stranded cDNA was then synthesized using the RNA template. Sequencing was done using a 3730xl DNA Analyzer.<em> </em>In the present study, c.A1459G (p.T487A) in Exon 5 in the DAXX gene was detected in Rin-5F cell lines, one of the X-ray-induced insulinomas. An NCBI homology search reveals that the 487 amino acid site in rats is the 497 amino acid of humans, based on the genomic cDNA homology between rats and humans. In humans, the COSMIC database suggests that mutations involving 497 amino acids have not been detected in all human cancers. However, the mutation of 496 amino acids was detected in human stomach and colon cancers. This is the first account of the DAXX gene's state in a cell line created by exposure to X-rays. This may point to the need for additional data and research on unique gene alterations involved in the development of X-ray-induced insulinoma tumors.

Target-capture full-length double-stranded cDNA long-read sequencing through Nanopore revealed novel intron retention in patient with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a relatively common autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms caused by loss-of-function mutation of either TSC1 or TSC2. The genetic diagnosis of inherited diseases, including TSC, in the clinical field is widespread using next-generation sequencing. The mutations in protein-coding exon tend to be verified because mutations directly cause abnormal protein. However, it is relatively difficult to verify mutations in the intron region because it is required to investigate whether the intron mutations affect the abnormal splicing of transcripts. In this study, we developed a target-capture full-length double-stranded cDNA sequencing method using Nanopore long-read sequencer (Nanopore long-read target sequencing). This method revealed the occurrence of intron mutation in the TSC2 gene and found that the intron mutation produces novel intron retention splicing transcripts that generate truncated proteins. The protein-coding transcripts were decreased due to the expression of the novel intron retention transcripts, which caused TSC in patients with the intron mutation. Our results indicate that Nanopore long-read target sequencing is useful for the detection of mutations and confers information on the full-length alternative splicing of transcripts for genetic diagnosis.

RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples.

Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol, the RAPIDprep assay, with the aim of providing a cause-agnostic laboratory diagnosis of infection within 24 h of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing, with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities, and extraction kits using a single workflow without input nucleic-acid quantification or quality assessment. Furthermore, we demonstrated the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPIDprep assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations.

Identification and genetic characterisatin of cathepsin L in Demodex.

Owing to difficulties in obtaining functional gene sequences, molecular pathogenic mechanisms in Demodex have been understudied. In this study, overlap extension PCR was used to obtain the sequences of cathepsin L (CatL), a pathogenicity-related gene, to provide a foundation for subsequent functional research. Demodex folliculorum and Demodex brevis mites were obtained from the face skin of Chinese individuals, and Demodex canis mites were isolated from the skin lesions of a dog. RNA was extracted and used to synthesise double-stranded cDNA. PCR amplification, cloning, sequencing, and bioinformatics analysis of CatL were performed. CatL gene sequences of 1005, 1008, and 1008bp were successfully amplified for D. brevis, D. folliculorum, and D. canis, respectively. These sequences showed 99.9 or 100% identity with templates previously obtained by RNA-seq. The Maximum Likelihood (ML) phylogenetic tree showed that D. folliculorum clustered with D. canis first, then with D. brevis, and finally with other Acariformes mite species. The three Demodex species had nine similar motifs to those of Sarcoptes scabies, Dermatophagoides pteronyssinus, and Dermatophagoides farinae, and motifs 10-13 were valuable for identification. CatL proteins of Demodex species were predicted to be approximately 38kDa, be located in lysosomes, have a signal peptide but no transmembrane region, and have two functional domains, I29 and Pept_C1. However, interspecific differences were observed in secondary and tertiary protein structures. In conclusion, we successfully obtained CatL sequences of three Demodex species by overlap extension PCR, which creates conditions for further pathogenic mechanism studies.

Analysis of Some Putative Novel Peptides from Iranian Scorpion Venom Glands, Hemiscorpius lepturus, Using cDNA Library Construction

Background: To date, more than 300,000 distinct peptides have been identified in scorpion venom. However, only a limited number of these peptides have been described. Objectives: We characterized some putative peptides from the venom gland cDNA library of the Iranian yellow scorpion Hemiscorpius lepturus. Methods: Total RNA was extracted from yellow Iranian scorpion glands. Single-stranded cDNA (sscDNA) and double-stranded cDNA (dscDNA) were synthesized by polymerase chain reaction (PCR). A cDNA library was achieved by inserting dscDNA into a special vector and subsequently transformed to chemically competent Escherichia coli as a host. The library was screened by culturing the liquid library on Lysogeny broth (LB)-agar plates. Analysis of positive clones was performed by plasmid extraction and the sequencing of the inserts. Finally, all cDNA sequences were analyzed and characterized by bioinformatics software. Results: One hundred colonies were randomly analyzed. Eighty-nine cDNA sequences had acceptable quality for bioinformatics analysis. Five sequences were selected for further analysis. The peptides related to these sequences were divided into two groups, non-disulfide bridge peptides (NDBP) and disulfide bridge peptides (DBP), the application of which in health and medical issues has been suggested. Conclusions: The data obtained in this study may be an important resource for further in vivo and in vitro functional assays to identify valuable therapeutic peptides.

A Universal, Single-Primer Amplification Protocol to Perform Whole-Genome Sequencing of Segmented dsRNA Avian Orthoreoviruses.

The Reoviridae family represents the largest family of double-stranded RNA viruses, and members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, and plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals and birds. Genomic characterization of reoviruses has not been adopted on a large scale because of the complexity of obtaining sequences for all 10 segments. In this study, we develop a time-efficient and practical method to enrich reovirus sequencing reads from isolates that allows for full-genome recovery using a single-primer amplification method coupled with next-generation sequencing. We refer to this protocol as reovirus-single-primer amplification (R-SPA). Our results demonstrate that most of the genes are covered with at least 500 reads per base space. Furthermore, R-SPA covers both the 5' and 3' ends of each reovirus genes. In summary, this study presents a universal and fast amplification protocol that yields sufficient double-stranded cDNA and facilitates and expedites the whole-genome sequencing of reoviruses.

A Modified Method of Total RNA Isolation for Mango Leaf Tissues

Complex metabolic components in mango leaves lead to high difficulty in RNA extraction. In order to improve the quality of total RNA extraction, on the basis of the existing methods reported in the literature, an RNA extraction method combining acetone washing liquid nitrogen abrasive material with 0.3 mol/L lithium chloride (LiCl) solution and 2 times the volume of anhydrous ethanol as the second precipitator was developed. The experimental results showed that the improved method significantly reduced the amount of impurity precipitation in the RNA extraction process. The electrophoretic bands of total RNA extracted were complete. The absorbance ratio of OD 260/280  was about 1.9, and the average was 329.8 g/g FW±11.2 g/g FW. Further validation experiments of RNA reverse transcription and the polymorphic amplification of double-stranded cDNA related sequences (RAP-PCR) obtained clear polymorphic bands, showing that the total RNA from mango leaves had high quality and was suitable for molecular biology experiments.

548. Target Enriched NGS Reveals Wide Breadth of Viruses Causing Acute Undifferentiated Fever in Thailand

Abstract Background Despite extensive laboratory testing, infectious agents were not detected in approximately 50% of patients hospitalized for acute undifferentiated febrile illnesses (AUFI) at Siriraj Hospital, Bangkok, Thailand. Unbiased Metagenomic Next Generation Sequencing (mNGS) enables detection of any microbe present in patient samples. Target enrichment for viruses represents a highly sensitive and cost-effective approach for overcoming host background in clinical specimens. Plasma collected from 800 patients with undiagnosed AUFI between 2013-2020 were analyzed by mNGS coupled to target enrichment to identify known and novel viruses. Methods Plasma was pre-treated with benzonase before extraction on an Abbott m2000 instrument. mNGS libraries were prepared from double-stranded cDNA with Illumina Nextera XT reagents on an epMotion then combined in pools of 24 and hybridized to Comprehensive Viral Research Panel (CVRP; Twist Biosciences) probes covering >15,000 strains of vertebrate viruses. Captured viral sequences were amplified, quantified, and sequenced together on a MiSeq. Reads were taxonomically classified by the SURPI pipeline and aligned in CLC Bio Genomics Workbench software. Results CVRP method optimization enabled sequencing of 24-48 libraries per MiSeq run, for which >50% genome coverage was obtained with model viruses spiked into clinical specimens at 1000 cp/ml. This approach revealed an array of >24 different viruses found in 26% of samples. Dengue was the most prevalent at 7.4%, with all four genotypes detected. Other common causes of AUFI such as Chikungunya, HIV-1, HAV, HBV, HCV, and CMV were present in 1.5-2.5% of cases. Less prevalent (< 1%) infections included HEV, HSV-2, EBV, HHV-6, Enterovirus B&D and Parvovirus B19. We also encountered sporadic cases of Measles, Cardiovirus, West Nile, Rotavirus, Picobirnavirus, Polyomavirus 4&5, Kubovirus, and Rabies lyssavirus. Conclusion Target capture successfully demonstrated that viruses were important etiologies for unresolved cases of AUFI in Thailand. This data has led to a better understanding of the epidemiology of this clinical syndrome and has implications for proper management of AUFI, including lower rates of unnecessary testing and antimicrobial use. Disclosures Julie Yamaguchi, BS, Abbott Labs: Employee Michael G. Berg, PhD, Abbott Labs: Employee Pakpoom Phoompoung, MD, Abbott Labs: Grant/Research Support|Abbott Labs: Employee Jenna Malinauskas, MS, Abbott Labs: Employee Gavin Cloherty, PhD, Abbott: Employee|Abbott: Stocks/Bonds|Abbott Labs: Employee Yupin Suputtamongkol, MD, Abbott Labs: Grant/Research Support.

Rapid and sensitive single-cell RNA sequencing with SHERRY2

BackgroundPrevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult.ResultsBased on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods.ConclusionsSHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.

Development of Multi-Epitopes Vaccine against Human Papilloma Virus16 Using the L1 and L2 Proteins as Immunogens

Background: Human papillomavirus 16 (HPV16) is a small non-enveloped DNA virus is belonging to Papillomaviridae. It usually causes warts and about 60% of cancer diseases. HPV16 genome consists of double-stranded cDNA of six early and two late proteins. This study attempted to design safe and efficient multi epitopes vaccine from structural proteins (L1 and L2) by using various immunoinformatic databases. The results demonstrated that the predicted vaccine comprised of 408aa and validated in terms of antigenicity, allergenicity, toxicity and stability by putting all critical parameters into consideration. The physiochemical properties displayed isoelectric point (pl) of 10.37. The instability index (II) was 33.6 categorizing vaccine as stable. The aliphatic index was 63.24 and the GRAVY was −0.652 demonstrating the hydrophilicity of the vaccine. Vaccine structures were predicted, refined and validated. Stability of the vaccine was assessed through Ramachandan plot and further assessed by ProSA server. Vaccine solubility was higher than the solubility of E. coli proteins indicating that the vaccine was soluble. Disulfide engineering increased the vaccine stability by substituting the unstable residues with cysteine residues. Vaccine-TLR4 receptor docking resulted in attractive binding energy of –1274.1 kcal/mol and –1450.4kcal/mol for chain A and chain B of the receptor respectively. Reverse transcription of the vaccine protein into a DNA sequence was performed and cloned into a pET30a (+) vector to confirm the clonability of the sequence during microbial expression. Taken together, the vaccine potentially induced immune responses and thus was suitable as a vaccine to combat HPV16 disease. Nonetheless, the efficiency of vaccines must be approved by in vitro and in vivo immunological analysis.

Transcriptome-wide measurement of poly(A) tail length and composition at subnanogram total RNA sensitivity by PAIso-seq.

Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAIso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains ~0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAIso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is ~14.5 h for PAIso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis.

Poly(A) capture full length cDNA sequencing improves the accuracy and detection ability of transcript quantification and alternative splicing events

The full-length double-strand cDNA sequencing, one of the RNA-Seq methods, is a powerful method used to investigate the transcriptome status of a gene of interest, such as its transcription level and alternative splicing variants. Furthermore, full-length double-strand cDNA sequencing has the advantage that it can create a library from a small amount of sample and the library can be applied to long-read sequencers in addition to short-read sequencers. Nevertheless, one of our previous studies indicated that the full-length double-strand cDNA sequencing yields non-specific genomic DNA amplification, affecting transcriptome analysis, such as transcript quantification and alternative splicing analysis. In this study, it was confirmed that it is possible to produce the RNA-Seq library from only genomic DNA and that the full-length double-strand cDNA sequencing of genomic DNA yielded non-specific genomic DNA amplification. To avoid non-specific genomic DNA amplification, two methods were examined, which are the DNase I-treated full-length double-strand cDNA sequencing and poly(A) capture full-length double-strand cDNA sequencing. Contrary to expectations, the non-specific genomic DNA amplification was increased and the number of the detected expressing genes was reduced in DNase I-treated full-length double-strand cDNA sequencing. On the other hand, in the poly(A) capture full-length double-strand cDNA sequencing, the non-specific genomic DNA amplification was significantly reduced, accordingly the accuracy and the number of detected expressing genes and splicing events were increased. The expression pattern and percentage spliced in index of splicing events were highly correlated. Our results indicate that the poly(A) capture full-length double-strand cDNA sequencing improves transcript quantification accuracy and the detection ability of alternative splicing events. It is also expected to contribute to the determination of the significance of DNA variants to splicing events.

FC047: RNA Sequencing of Microdissected Kidney Biopsies from IGA Nephropathy Patients

Abstract BACKGROUND AND AIMS As the most common glomerulonephritis globally, mainly affecting younger adults and one of the most common causes of kidney failure [1], IgA Nephropathy (IgAN) poses an immense impact both on the individual and on the societal level. Here, we aimed to gain further molecular insight into the high interindividual variability of the clinical picture and disease progression by linking transcriptional profiling of IgAN-kidneys to clinical and histopathological data. METHOD Kidney biopsies from patients with histopathologically verified IgAN/IgA vasculitis (n = 84) (IgAV) [57 males, median (range) age of 39 (4–89) years] and 11 living donors (LD) [six males, median (range) age of 37 (30–65) years] were manually microdissected into glomerular and tubulointerstitial fractions. RNA was extracted with RNeasy lipid tissue mini kit (Qiagen, Valencia, CA, US) and evaluated using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, US). mRNA purification, conversion to cDNA, fragmentation and double-stranded cDNA synthesis, amplification and clean-up were performed using Illumina Stranded mRNA prep ligation protocol (Illumina Inc). Paired-end RNA sequencing was performed in three different batches (Illumina Novaseq 6000). Data pre-processing was done using Trim Galore (v.0.6.4). Reads were aligned to the Ensembl GRCh38 reference genome using STAR (v2.6.1d). Gene counts were obtained using featureCounts (v1.5.1). Quality control was made using MultiQC (v.1.7). Comparisons between groups were performed using Bioconductor package DESeq2 (v1.22.2) and gene set enrichment analysis using Bioconductor package fgsea. The biopsies were evaluated by experienced renal pathologists using the Oxford classification [2] and the Banff classifications [3]. Clinical data at time of biopsy and 5-year follow-up was retrieved from patients’ files. Co-morbidity- and mortality data was extracted from the Swedish renal registry up to 19 years after the biopsy. RESULTS Principal Component Analysis showed clear separation between diseased and LD kidney transcriptomes, as well as between glomerular and tubulointerstitial fractions. Top upregulated genes in glomeruli were associated with complement activation and fibrosis. In tubulointerstium, top genes were related to the immune system, including chemokines. Gene ontology enrichment analysis highlighted immune response and complement activation in both compartments. Additionally, cell membrane and mitochondrial activity were enriched in tubulointerstium. Linking bioinformatic results to clinical data at the time of biopsy, progress over time as well as to histopathological data in accordance with the Oxford classification system [2] is ongoing. CONCLUSION This is, to our knowledge, the most comprehensive RNA sequencing performed on paired glomerular and tubulointerstitial tissue from patients with IgAN/IgAV. Initial bioinformatical analyses highlights biologically relevant processes involving different parts of the immune system. Our project has the potential to identify molecular processes associated with rapid disease progression and specific histopathological characteristics. This enables identification of patients with a more aggressive disease and individualized treatment depending on molecular profile, which would improve kidney survival and quality of life for patients suffering from IgAN/IgAV.

Developing RNA‐Seq and ChIP‐Seq Pipelines on an Oxford Nanopore Technologies Platform to Study Notch1 Transcriptional Dynamics

Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface which is cleaved resulting in the release of the intracellular domain (NICD). The NICD enters the nucleus and complexes with CBF1 and Mastermind (MAM) to activate target genes. CBF1 recognizes the binding motif RTGRGAR (R=G or A), and this is referred to an NRE (Notch Response Element). Recent studies have shown that a paired CBF1 binding site is also important for target gene activation. While individual genes regulated by SPS are known, the extent to which an SPS vs individual NREs are responsible for Notch‐mediated transcription on a genomic level remains an active area of research. It is hypothesized that the majority of Notch‐mediated transcription occurs as a result of a dimeric Notch complex in the promoters or enhancers of transcribed genes. Recently, high‐throughput techniques have allowed the study of Notch complex assembly and their effects on transcription. Here, we use an Oxford Nanopore Platform was used.To identify genes regulated by Notch transcriptional complexes, RNA‐Seq was performed. Total RNA was isolated from SUPT1 cells treated with 5 µM of the Notch inhibitor DAPT for 24 hours, or DMSO as a control. Poly‐A RNA was purified using an oligo‐dT column and double‐stranded cDNA generated. PCR adapters were nonspecifically ligated to cDNA and amplified to increase library yield. Sequencing was performed using an Oxford Nanopore Minion, and results were mapped to the human reference genome to identify genes significantly downregulated when Notch was inhibited. To identify NREs and SPS in the regulatory elements of these genes, ChIP‐Seq was performed for Notch. ChIP was performed in DAPT and DMSO treated cells, and ChIP DNA used to prepare a sequencing library as above. Sequenced fragments were aligned to a known regulatory elements dataset, and genes with significant Notch binding identified.RNA‐Seq data shows significant downregulation of known Notch targets genes, such as Hes1, Hey1, and Hes4. Sequenced mRNA fragments, however, averaged 1kb suggesting size selectivity during the purification or amplification steps of library preparation. Additionally, underrepresentation of other Notch known targets such as Hes5 may suggest that computational stringency may have to be adjusted. We are currently pursuing ChIP‐Seq experiments that will be presented at the conference to identify the locations of SPS in the genome. Identified SPS in regulatory elements will then be correlated with the genes they activate, and the downstream effects on transcription analyzed. It is expected that a large number of genes that are inactivated under Notch inhibition conditions will exhibit a SPS in a corresponding regulatory element.

A comparison of mRNA sequencing (RNA-Seq) library preparation methods for transcriptome analysis

BackgroundmRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis.ResultsWe observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated.ConclusionsTruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime with regards to the detected number of transcripts and splicing events among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.

Gene expression profile in functioning and non-functioning nodules of autonomous multinodular goiter from an area of iodine deficiency: unexpected common characteristics between the two entities

PurposeToxic multinodular goiter is a heterogeneous disease associated with hyperthyroidism frequently detected in areas with deficient iodine intake, and functioning and non-functioning nodules, characterized by increased proliferation but opposite functional activity, may coexist in the same gland. To understand the distinct molecular pathology of each entity present in the same gland, the gene expression profile was evaluated by using the Affymetrix technology.MethodsTotal RNA was extracted from nodular and healthy tissues of two patients and double-strand cDNA was synthesized. Biotinylated cRNA was obtained and, after chemical fragmentation, was hybridized on U133A and B arrays. Each array was stained and the acquired images were analyzed to obtain the expression levels of the transcripts. Both functioning and non-functioning nodules were compared versus healthy tissue of the corresponding patient.ResultsAbout 16% of genes were modulated in functioning nodules, while in non-functioning nodules only 9% of genes were modulated with respect to the healthy tissue. In functioning nodules of both patients and up-regulation of cyclin D1 and cyclin-dependent kinase inhibitor 1 was observed, suggesting the presence of a possible feedback control of proliferation. Complement components C1s, C7 and C3 were down-regulated in both types of nodules, suggesting a silencing of the innate immune response. Cellular fibronectin precursor was up-regulated in both functioning nodules suggesting a possible increase of endothelial cells. Finally, Frizzled-1 was down-regulated only in functioning nodules, suggesting a role of Wnt signaling pathway in the proliferation and differentiation of these tumors. None of the thyroid-specific gene was deregulated in microarray analysis.ConclusionIn conclusion, the main finding from our data is a similar modulation for both kinds of nodules in genes possibly implicated in thyroid growth.

Amplification of a minimally biased antibody repertoire for in vitro display using a universal primer-based amplification method

Immune hosts are valuable sources for antibody discovery. To construct in vitro display antibody libraries from immune repertoires, singleplex or multiplex PCR amplification were employed using primers targeting multiple immunoglobulin genes. However, during this process, the B cell receptor repertoire is distorted due to interactions between multiple target genes and primers. To minimize this alternation, we devised a new method for harvesting immunoglobulin genes and tested its performance in rabbit variable heavy chain (VH) and variable kappa light chain (VK) genes. Double-stranded cDNA was synthesized using primers containing V/J gene–specific regions and universal sequence parts for in vitro display. VH and VK gene libraries were obtained through subsequent PCR amplification using primers with universal sequences. Next-generation sequencing analysis confirmed that universal PCR libraries had more diverse VH and VK clonotypes, and a less biased clonal distribution, than conventional singleplex or multiplex gene-specific PCR libraries.