cDNA Copy Research Articles

Simultaneous detection of dengue virus serotypes in a dual-serotype-detection nucleic acid based lateral flow assay.

Dengue virus (DENV) is an important arthropod-borne viral disease, with four antigenically and genetically diverse serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). Timely and accurate diagnosis of dengue virus serotypes is crucial for the management of outbreaks. This study focussed on the development of a RT-PCR based lateral flow strip assay to detect DENV serotypes in a dual detection manner without using gel electrophoresis. The assay uses anti-biotin/streptavidin colloidal gold conjugates with fluorescent/enzymatic tagged DENV serotype specific antibodies for the direct detection of DENV infected serum samples on a nitrocellulose membrane using biotin-BSA as control line. The detection limit of the assay was up to 10 copies of cDNA for DENV-1 and 100 copies for DENV-2, DENV-3, and DENV-4. In house evaluation of DENV LFIA demonstrated 100 % sensitivity in all the serotypes compared to conventional RT-PCR, 100 % specificity for DENV-1, DENV-2, DENV-3, and 95 % specificity for DENV-4 detection. DENV serotyping was assessed in a dual detection manner (DENV-1/DENV-3 and DENV-2/DENV-4 at two test lines) on the strip. The limitation of the assay is the requirement of PCR for initial amplification and confirmation of individual serotype in case of DENV-1/DENV-3 and DENV-2/DENV-4 detection, besides the field evaluation of the assay detected DENV-2 and DENV-3 serotypes, and no other serotype was detected in line with RT-PCR findings.

Evaluation of adropin, fibroblast growth factor-1 (FGF-1), and Toll-like receptor-1 (TLR1) biomarkers in patients with inflammatory bowel disease: gene expression of TNF-α as a marker of disease severity

BackgroundInflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder of unknown etiology and unpredictable course. The aim of the work was to assess the levels of adropin, fibroblast growth factor-1 (FGF-1), and Toll-like receptor-1 (TLR1) biomarkers in IBD patients compared to controls and evaluate the gene expression of TNF-α as a marker of disease severity.MethodsAdropin, fasting serum FGF-1 levels, TLR1, and TNF-α were measured in 60 IBD patients. They were also compared with 58 healthy controls matching age and gender. Moreover, the blood cells cDNA copy number of TNF-α were determined as a marker of severity.ResultsAdropin and TLR1 levels were significantly lower in patients than controls. FGF-1 was reduced but not statistically significant. The expression of TNF-α gene in the IBD patients was significantly increased (42%) in comparison with control samples (P < 0.001).ConclusionsAdropin, IGF-I, and Toll-like receptor-1 biomarkers may have a role in the intricate pathophysiology of IBD and may possibly operate as predictors of disease activity. Thus, they may be therapeutic targets for IBD. Moreover, the expression of TNF-α gene can be used as a marker of severity.

Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis.

Nanopore direct RNA sequencing (DRS) enables measurements of RNA modifications. Modification-free transcripts are a practical and targeted control for DRS, providing a baseline measurement for canonical nucleotides within a matched and biologically derived sequence context. However, these controls can be challenging to generate and carry nanopore-specific nuances that can impact analysis. We produced DRS datasets using modification-free transcripts from in vitro transcription (IVT) of cDNA from six immortalized human cell lines. We characterized variation across cell lines and demonstrated how these may be interpreted. These data will serve as a versatile control and resource to the community for RNA modification analysis of human transcripts.

Genomic surveillance of SARS-CoV-2 using long-range PCR primers.

Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). In this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants.

An ultra high-throughput, massively multiplexable, single-cell RNA-seq platform in yeasts.

Yeasts are naturally diverse, genetically tractable, and easy to grow such that researchers can investigate any number of genotypes, environments, or interactions thereof. However, studies of yeast transcriptomes have been limited by the processing capabilities of traditional RNA sequencing techniques. Here we optimize a powerful, high-throughput single-cell RNA sequencing (scRNAseq) platform, SPLiT-seq (Split Pool Ligation-based Transcriptome sequencing), for yeasts and apply it to 43,388 cells of multiple species and ploidies. This platform utilizes a combinatorial barcoding strategy to enable massively parallel RNA sequencing of hundreds of yeast genotypes or growth conditions at once. This method can be applied to most species or strains of yeast for a fraction of the cost of traditional scRNAseq approaches. Thus, our technology permits researchers to leverage "the awesome power of yeast" by allowing us to survey the transcriptome of hundreds of strains and environments in a short period of time and with no specialized equipment. The key to this method is that sequential barcodes are probabilistically appended to cDNA copies of RNA while the molecules remain trapped inside of each cell. Thus, the transcriptome of each cell is labeled with a unique combination of barcodes. Since SPLiT-seq uses the cell membrane as a container for this reaction, many cells can be processed together without the need to physically isolate them from one another in separate wells or droplets. Further, the first barcode in the sequence can be chosen intentionally to identify samples from different environments or genetic backgrounds, enabling multiplexing of hundreds of unique perturbations in a single experiment. In addition to greater multiplexing capabilities, our method also facilitates a deeper investigation of biological heterogeneity, given its single-cell nature. For example, in the data presented here, we detect transcriptionally distinct cell states related to cell cycle, ploidy, metabolic strategies, and so forth, all within clonal yeast populations grown in the same environment. Hence, our technology has two obvious and impactful applications for yeast research: the first is the general study of transcriptional phenotypes across many strains and environments, and the second is investigating cell-to-cell heterogeneity across the entire transcriptome.

Human glioma malignancy grade and migratory capacity depending on expression of GDNF isoforms in vitro

To assess the expression of mRNA splice variants of GDNF in glioma cell cultures with various malignancy grades (I-IV) and degrees of migration. In this study, αGDNF and βGDNF expression was analyzed in 15 human glioma cell cultures using Southern blot hybridization of GDNF cDNA and reverse transcription with PCR to amplify splice variants of GDNF mRNA. The highest expression of αGDNF and βGDNF isoforms was observed in cell cultures of human gliomas with extensive migratory activity. Low βGDNF expression without αGDNF expression is typical only for gliomas with low migratory activity. In addition, we found additional patterns of mRNA expression that have not been previously described. The relationship between GDNF and malignancy grade is unclear. Nevertheless, GDNF expression is higher in glioblastomas. Overall GDNF expression is increased in glioma cells with high migration activity. At the same time, αGDNF and βGDNF isoforms demonstrate higher expression in actively migrating cells that can indicate their participation in regulation of tumor migration properties. No αGDNF expression with simultaneous low βGDNF expression may be a prognostic sign of low migration activity of human glioma cells.

Molecular and functional characterization of two granulocyte colony stimulating factors in goldfish (Carassius auratus L.).

Granulocyte colony-stimulating factor (GCSF) is a member of the hematopoietic growth factor family that acts primarily on neutrophils and neutrophilic precursors to promote cell proliferation and differentiation. Although multiple GCSF genes have been found in teleosts, knowledge of their functions during fish hematopoietic development is still limited. Here, we report for the first time the molecular and functional characterization of two goldfish GCSFs (gfGCSF-a and gfGCSF-b). The open reading frame (ORF) of the gfGCSF-a and gfGCSF-b cDNA transcript consisted respectively of 624bp and 678bp with its ORF encoding 207 and 225 amino acids (aa), with a 17 aa signal peptide for each gene and a conserved domain of the IL-6 superfamily. Treatment of goldfish head kidney leukocytes (HKLs) with LPS increased gfGCSF-a and gfGCSF-b mRNA expression levels, also exposure of HKLs to either heat-killed or live A. hydrophila, induced transcriptional upregulation of gfGCSF-a and gfGCSF-b levels. Recombinant gfGCSF-a and gfGCSF-b protein (rgGCSF-a and rgGCSF-b) induced a dose-dependent production of TNFα and IL-1β from goldfish neutrophils. In vitro experiments showed rgGCSF-a and rgGCSF-b differentially promoted the proliferation and differentiation of leukocytes in goldfish. Furthermore, treatment of HKLs with rgGCSF-a showed significant upregulation of mRNA levels of the hematopoietic transcription factor GATA2, Runx1, MafB, and cMyb, while gfGCSF-b induces not only all four transcriptional factors mentioned above but also CEBPα. Our results indicate that goldfish GCSF-a and GCSF-b are important regulators of neutrophil proliferation and differentiation, which could stimulate different stages and lineages of hematopoiesis.

Immunohistochemical and Ultrastructural Imaging of the SARS-CoV-2 Coronavirus in Lung, Lymph Node, and Kidney Tissues of Deceased Patients

The objective: immunohistochemical and electron microscopic imaging of the SARS-CoV-2 coronavirus in lung, lymph nodes and kidney tissues of patients who died of COVID-19.Subjects and Methods. For immunohistochemical tests, specimens of sections of formalin-fixed and paraffin-embedded tissues of the lungs, lymph nodes and kidneys of patients who died from COVID-19 were used. Quantitative assessment of the SARS-CoV-2 viral load level was carried by the original RT qPCR and calculated by the formula: SARS-CoV-2 copy number/ABL1 х 100 copies, expressed as the ratio of the true number of SARS-CoV-2 cDNA copies per 100 copies cDNA of the ABL1 gene. For morphological tests, samples of native lung, lymph node and kidney tissues were taken.Results. Immunohistochemical and electron microscopic tests revealed particles of the SARS-CoV-2 coronavirus in the cytoplasm of endothelial cells of air-blood barrier of the lungs, the vascular glomerulus of the kidneys, in the cytoplasm of macrophages of the lymph node, and also in cytoplasm of lymphocytes in the lumen of lung capillaries.

Development of an AAV-CRISPR-Cas9-based treatment for dominant cone-rod dystrophy 6

Cone-rod dystrophy 6 (CORD6) is caused by gain-of-function mutations in the GUCY2D gene, which encodes retinal guanylate cyclase-1 (RetGC1). There are currently no treatments available for this autosomal dominant disease, which is characterized by severe, early-onset visual impairment. The purpose of our study was to develop an adeno-associated virus (AAV)-CRISPR-Cas9-based approach referred to as "ablate and replace" and evaluate its therapeutic potential in mouse models of CORD6. This two-vector system delivers (1) CRISPR-Cas9 targeted to the early coding sequence of the wild-type and mutant GUCY2D alleles and (2) a CRISPR-Cas9-resistant cDNA copy of GUCY2D ("hardened" GUCY2D). Together, these vectors knock out ("ablate") expression of endogenous RetGC1 in photoreceptors and supplement ("replace") a healthy copy of exogenous GUCY2D. First, we confirmed that ablation of mutant R838S GUCY2D was therapeutic in a transgenic mouse model of CORD6. Next, we established a proof ofconcept for "ablate and replace" and optimized vector dosesinGucy2e+/-:Gucy2f-/- and Gucy2f-/- mice, respectively. Finally, we confirmed that the "ablate and replace" approach stably preserved retinal structure and function in a novel knockin mouse model of CORD6, the RetGC1 (hR838S, hWT) mouse. Taken together, our results support further development of the "ablate and replace" approach for treatment of CORD6.

Smar2C2: A Simple and Efficient Protocol for the Identification of Transcription Start Sites.

Promoters and the noncoding sequences that drive their function are fundamental aspects of genes that are critical to their regulation. The transcription preinitiation complex binds and assembles on promoters where it facilitates transcription. The transcription start site (TSS) is located downstream of the promoter sequence and is defined as the location in the genome where polymerase begins transcribing DNA into RNA. Knowing the location of TSSs is useful for annotation of genes, identification of non-coding sequences important to gene regulation, detection of alternative TSSs, and understanding of 5' UTR content. Several existing techniques make it possible to accurately identify TSSs, but are often difficult to perform experimentally, require large amounts of input RNA, or are unable to identify a large number of TSSs from a single sample. Many of these protocols take advantage of template switching reverse transcriptases (TSRTs), which reliably place an adaptor at the 5' end of a first strand synthesis of cDNA. Here, we introduce a protocol that exploits TSRT activity combined with rolling circle amplification to identify TSSs with several unique advantages over existing methods. Sequence adaptors are placed on the 5' and 3' end of the full-length cDNA copy of a transcript. A splint compatible with those adaptors is then used to circularize the full-length cDNA. Linear DNA containing concatemers of the cDNA are generated using rolling circle amplification, and a sequencing library is formed by fragmenting the concatemers. This protocol is straightforward to execute, requiring limited bench time with relatively stable reagents. Using extremely low amounts of RNA input, this protocol produces large numbers of accurate, deduplicated TSSs genome wide. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Splint generation Basic Protocol 2: RNA extraction Basic Protocol 3: cDNA synthesis Basic Protocol 4: cDNA circularization and amplification Basic Protocol 5: Library generation.

A Computational Workflow for Analysis of 3' Tag-Seq Data.

RNA-sequencing (RNA-seq) is a gold-standard method to profile genome-wide changes in gene expression. RNA-seq uses high-throughput sequencing technology to quantify the amount of RNA in a biological sample. With the increasing popularity of RNA-seq, many variations on the protocol have been proposed to extract unique and relevant information from biological samples. 3' Tag-Seq (also called TagSeq, 3' Tag-RNA-Seq, and Quant-Seq 3' mRNA-Seq) is one RNA-seq variation where the 3' end of the transcript is selected and amplified to yield one copy of cDNA from each transcript in the biological sample. We present a simple, easy-to-use, and publicly available computational workflow to analyze 3' Tag-Seq data. The workflow begins by trimming sequence adapters from raw FASTQ files. The trimmed sequence reads are checked for quality using FastQC and aligned to the reference genome, and then read counts are obtained using STAR. Differential gene expression analysis is performed using DESeq2, based on differential analysis of gene count data. The outputs of this workflow are MA plots, tables of differentially expressed genes, and UpSet plots. This protocol is intended for users specifically interested in analyzing 3' Tag-Seq data, and thus normalizations based on transcript length are not performed within the workflow. Future updates to this workflow could include custom analyses based on the gene counts table as well as data visualization enhancements. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Running the 3' Tag-Seq workflow Support Protocol: Generating genome indices.

Functional verification and characterization of a type-III geranylgeranyl diphosphate synthase gene from Sporobolomyces pararoseus NGR.

Carotenoids, a group of natural pigments, have strong antioxidant properties and act as precursors to vitamin A, which have garnered attention from industry and researchers. Sporobolomyces pararoseus represents a hyper-producer of carotenoids, mainly including β-carotene, torulene, and torularhodin. Geranylgeranyl diphosphate synthase (GGPPS) is regarded as a key enzyme in the carotenoid biosynthesis pathway. However, the precise nature of the gene encoding GGPPS in S. pararoseus has not been reported yet. Here, we cloned a cDNA copy of the GGPPS protein-encoding gene crtE from S. pararoseus NGR. The crtE full-length genomic DNA and cDNA are 1,722 and 1,134 bp, respectively, which consist of 9 exons and 8 introns. This gene encodes 377 amino acids protein with a predicted molecular mass of 42.59 kDa and a PI of 5.66. Identification of the crtE gene encoding a functional GGPPS was performed using heterologous complementation detection in Escherichia coli. In vitro enzymatic activity experiments showed that CrtE utilized farnesyl diphosphate (FPP) as an allylic substrate for the condensation reaction with isopentenyl diphosphate (IPP), generating more of the unique product GGPP compared to other allylic substrates. The predicted CrtE 3D-model was analyzed in comparison with yeast GGPPS. The condensation reaction occurs in the cavity of the subunit, and three bulky amino acids (Tyr110, Phe111, and His141) below the cavity prevent further extension of the product. Our findings provide a new source of genes for carotenoid genetic engineering.

Melanopsin expression in the retinas of owls with different daily activity patterns

Melanopsin is a photopigment found in a subset of retinal ganglion cells that is responsible for generating a series of responses to light in organisms, such as circadian rhythm regulation, pupillary light reflex, and body temperature control. The role of each melanopsin gene in the vertebrate retina is still not fully elucidated and a diversity of expression patterns of this photopigment can be observed in retinas of different vertebrate species. Owls are an excellent model for studying the role of melanopsin due to the diversity of species, which may present diurnal, nocturnal, or cathemeral habits. The purpose of this study was to characterize the expression of melanopsin genes in the retina of four different owl species from the Strigidae family (Athene cunicularia, Asio clamator, Glaucidium brasilianum, and Megascops choliba) through genetic and phylogenetic analysis. The specimens were euthanized, and the retinas were collected for RNA extraction and cDNA transcription. cDNA was used in the polymerase chain reaction (PCR) with subsequent sequencing to identify melanopsin genes expressed in the retina of owls. For the quantitative analysis of gene expression, real-time PCR was performed. The phylogenetic reconstruction was obtained by the maximum likelihood. The results showed that owls express both melanopsin genes, Opn4x and Opn4m, with different patterns of expression among the species. The expression of the Opn4x gene was two times higher in Asio clamator (nocturnal) compared to A. cunicularia (cathemeral), Glaucidium brasilianum (diurnal), and Megascops choliba (nocturnal). On the other hand, the expression of Opn4m was about two times lower in the cathemeral, A. cunicularia compared to the other three species. These results might indicate functional differences of the Opn4x and Opn4m genes among species, related to circadian rhythm regulation. Further investigation in owls at other times of the day will bring light to the circadian pattern of melanopsin expression in these species and its correlation with the different patterns of daily activity.

Whole-Genome and Long-Read Sequencing Identify a Novel Mechanism in RFC1 Resulting in CANVAS Syndrome.

Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) results from biallelic intronic pentanucleotide repeats in RFC1. We describe an adult male proband with progressive imbalance, cerebellar atrophy, somatosensory neuronopathy, and absence of peripheral vestibular function for whom clinical testing demonstrated a heterozygous RFC1 expansion consistent with an unaffected carrier. We performed whole-genome sequencing (WGS) on peripheral blood DNA samples from the proband and his unaffected mother. We performed DNA long-read sequencing and synthesized complementary DNA from RNA using peripheral blood from the proband. WGS confirmed the maternally inherited RFC1 expansion and identified a rare, nonsense RFC1 variant: c.C1147T; p.R383X in the proband but not the maternal DNA sample. RFC1 variants were confirmed in trans with long-read sequencing. Functional studies demonstrated the absence of complementary DNA (cDNA) transcript from the c.C1147T; p.R383X variant supporting nonsense-mediated decay of this transcript. We report an adult with CANVAS due to compound heterozygous pathogenic RFC1 variants: the pathogenic intronic pentanucleotide expansion confirmed in trans with a nonsense variant. This report represents a novel molecular mechanism for CANVAS. Sequencing for RFC1 should be considered for adults meeting clinical criteria for the CANVAS phenotype if only a heterozygous pathogenic RFC1 expansion is identified.

HIV-1 Infection of Long-Lived Hematopoietic Precursors In Vitro and In Vivo.

Latent reservoirs in human-immunodeficiency-virus-1 (HIV-1)-infected individuals represent a major obstacle in finding a cure for HIV-1. Hematopoietic stem and progenitor cells (HSPCs) have been described as potential HIV-1 targets, but their roles as HIV-1 reservoirs remain controversial. Here we provide additional evidence for the susceptibility of several distinct HSPC subpopulations to HIV-1 infection in vitro and in vivo. In vitro infection experiments of HSPCs were performed with different HIV-1 Env-pseudotyped lentiviral particles and with replication-competent HIV-1. Low-level infection/transduction of HSPCs, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPP), was observed, preferentially via CXCR4, but also via CCR5-mediated entry. Multi-lineage colony formation in methylcellulose assays and repetitive replating of transduced cells provided functional proof of susceptibility of primitive HSPCs to HIV-1 infection. Further, the access to bone marrow samples from HIV-positive individuals facilitated the detection of HIV-1 gag cDNA copies in CD34+ cells from eight (out of eleven) individuals, with at least six of them infected with CCR5-tropic HIV-1 strains. In summary, our data confirm that primitive HSPC subpopulations are susceptible to CXCR4- and CCR5-mediated HIV-1 infection in vitro and in vivo, which qualifies these cells to contribute to the HIV-1 reservoir in patients.

PB1791: ON THE ISSUE OF HOUSEKEEPING GENE PRIMER DESIGN FOR ADEQUATE ASSESSMENT OF THE EXPRESSION OF THE CHIMERIC TRANSCRIPTS.

Background: Chimeric transcripts, which are the markers of hematological malignancies, is often assessed by expression level relative to the control housekeeping gene. The critical step for the analysis is RNA isolation and subsequent cDNA synthesis. Routine RNA isolation methods do not guarantee the absence of genomic DNA impurities, which may interfere with cDNA amplification. This results in the bias in control gene expression and underestimation of the relative expression. The use of sophisticated RNA isolation procedures or treatment with RNase-free DNAses significantly complicates and increases the cost of analysis, and can also lead to degradation of target sequences by endogenous nucleases. Here we describe a simple and effective solution to this problem. Aims: To optimize the RT-PCR approach for quantitative assessment of the chimeric transcripts gene expression relative to the control ABL1 gene, for the insensitivity to genomic DNA impurities. Methods: Total RNA was isolated from peripheral blood leukocytes or bone marrow cells as described by Chomczynski (1987). RT-PCR was performed using reagents and primers from Synthol (Moscow, Russia). Results: For ABL1 mRNA amplification primers flanking 2 gene introns were designed (see Table 1). This precludes PCR amplification of the corresponding (139708bp long) genomic sequence. At the same time, the cDNA of this fragment is only 100 nucleotides long. The primers recommended by Europe Against Cancer Association (EAC) and widely used (including as part of commercial diagnostic kits) flank 687bp ABL1 gene DNA fragment. In this case, the admixture of genomic DNA is co-amplified with the cDNA transcript in PCR. We have compared CBFB::MYH11 chimeric transcript relative expression levels in patients with AML, obtained with ABL-EAC primers and those proposed by us (ABL-N). We have tested 40 cDNA samples obtained from diverse RNA isolation routines, including 8 archival samples stored in the freezer for more than 6 months. The threshold cycles of amplification (ΔCt) for the samples from freshly isolated RNA were mostly the same with ABL-EAC and ABL-N primers. For archival RNA, cDNA, and RNA isolated from frozen cells, ΔCt with ABL-N primers drops significantly while ΔCt with ABL-EAC primers is preserved. This may indicate degradation in archival RNA and cDNA samples, while the level of genomic DNA decreases slightly. Thus, usage of ABL-EAC primers may lead in some circumstances to a significant bias in chimeric transcript evaluation. While primers we offer (ABL-N) are still effective even in the cases with compromised sample quality. Image:Summary/Conclusion: Genomic DNA amplification does not tamper with fusion gene transcripts RT-PCR measurement due to the absence of an appropriate target in the genomic DNA. While primers for measuring housekeeping genes expression (even those widely used) may have these targets and therefore amplify genomic DNA along with cDNA. Here we propose primers for measuring ABL1 control gene expression allowing reliable calculation of chimeric transcripts relative expression, regardless of RNA sample purity, in contrast to primers recommended by the EAC.

Abstract 496: Novel sample isolation and immortalization method for DNA and RNA to extend translational research capabilities

Abstract Background: Translational cancer research is dependent on well annotated human samples, but scarcity of specimen DNA and RNA hinders research and novel diagnostic testing. We demonstrate a novel nucleic acid isolation and anchoring approach that facilitates uniform, consistent replication of the isolated and captured DNA and RNA. This approach allows for expanded investigations of precious samples and increases the value of biobanked material. Methods: Our simplSEQ replication methodology enzymatically adds homopolymeric tails to the 3’ end of DNA and RNA, with a terminal dideoxy biotin-modified NTP, to attach the nucleic acids to streptavidin beads. The 3’ immobilized nucleic acids are copied using complementary homopolymers. The simplSEQ advantage is that molecules of all sizes are attached and copied from their 3’ ends yielding cDNA copies that are full-length representations of the input. Various DNA samples were investigated to determine the characteristics of cDNA copies as well as stability of bound nucleic acids. Samples evaluated included Human 1,000 genome project, bacterial genomes, commercial DNA controls, and synthesized DNA molecules. The samples represent a range of DNA sizes, AT/GC content and Variant Allele Frequencies (VAF). NGS assays quantified the presence of full-length genomic sequences. Variant analysis determined accuracy of copies based on comparison with published data (NA12878). Synthetic DNA oligonucleotides representing regions in the human genome and constructed to enable molecular counting independent of adapter ligation efficiency. All sample types were replicated, sequenced, and analyzed for coverage and uniformity. This replication process was repeated multiple times. Results: Three replicates of each DNA type were attached to streptavidin beads and subjected to multiple rounds of the simplSEQ protocol. Analysis of overall coverage and variant calling for human NA12878 replicates compared to the platinum standard NA12878 genome show an average coverage ranging from 27x-32x with &amp;gt;99% correlation between replicates. Sensitivity ranging from 98.6%-99.5%, with a precision range from 96.0% to 98.0% for 6 replicates. Bacterial samples with coverage over 100x and &amp;gt;99% correlation. Synthetic DNA oligonucleotide pools with coverage over 10,000x and &amp;gt;99% correlation. Conclusion: Our novel enzymatic tailing methodology produces highly accurate copies of full-length nucleic acids. This technology obviates sample scarcity for genomic evaluations and allows for expanded hypothesis testing in translational research. This methodology also provides an option for extended storage of nucleic acids from which replicates can be repeatedly generated to allow for reinterrogation. Citation Format: John Christopher Spinosa, Brandon Young, Vincent Aguilar, Baiju Parikh. Novel sample isolation and immortalization method for DNA and RNA to extend translational research capabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 496.

Sequencing of individual barcoded cDNAs using Pacific Biosciences and Oxford Nanopore Technologies reveals platform-specific error patterns.

Long-read transcriptomics require understanding error sources inherent to technologies. Current approaches cannot compare methods for an individual RNA molecule. Here, we present a novel platform-comparison method that combines barcoding strategies and long-read sequencing to sequence cDNA copies representing an individual RNA molecule on both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). We compare these long-read pairs in terms of sequence content and isoform patterns. Although individual read pairs show high similarity, we find differences in (1) aligned length, (2) transcription start site (TSS), (3) polyadenylation site (poly(A)-site) assignment, and (4) exon–intron structures. Overall, 25% of read pairs disagree on either TSS, poly(A)-site, or splice site. Intron-chain disagreement typically arises from alignment errors of microexons and complicated splice sites. Our single-molecule technology comparison reveals that inconsistencies are often caused by sequencing error–induced inaccurate ONT alignments, especially to downstream GUNNGU donor motifs. However, annotation-disagreeing upstream shifts in NAGNAG acceptors in ONT are often confirmed by PacBio and are thus likely real. In both barcoded and nonbarcoded ONT reads, we find that intron number and proximity of GU/AGs better predict inconsistencies with the annotation than read quality alone. We summarize these findings in an annotation-based algorithm for spliced alignment correction that improves subsequent transcript construction with ONT reads.

Modes of Viroid Transmission.

Studies on the ways in which viroids are transmitted are important for understanding their epidemiology and for developing effective control measures for viroid diseases. Viroids may be spread via vegetative propagules, mechanical damage, seed, pollen, or biological vectors. Vegetative propagation is the most prevalent mode of spread at the global, national and local level while further dissemination can readily occur by mechanical transmission through crop handling with viroid-contaminated hands or pruning and harvesting tools. The current knowledge of seed and pollen transmission of viroids in different crops is described. Biological vectors shown to transmit viroids include certain insects, parasitic plants, and goats. Under laboratory conditions, viroids were also shown to replicate in and be transmitted by phytopathogenic ascomycete fungi; therefore, fungi possibly serve as biological vectors of viroids in nature. The term “mycoviroids or fungal viroids” has been introduced in order to denote these viroids. Experimentally, known sequence variants of viroids can be transmitted as recombinant infectious cDNA clones or transcripts. In this review, we endeavor to provide a comprehensive overview of the modes of viroid transmission under both natural and experimental situations. A special focus is the key findings which can be applied to the control of viroid diseases.

Identification of Viral Nervous Necrosis (VNN) in Grouper Fish (Epinephelus sp.) by Reverse Transcriptase-Polymerase Chain Reaction

Viral Nervous Necrosis (VNN) is a Nodaviridae virus-infected snapper and grouper fish, especially in the larval and seed stages targeted eye and nervous organ. Abnormal swimming was shown by fish behavior at the bottom pond. This study aimed to compare the identification of VNN through three different methods of RNA extraction from the organ target and amplification was carried out by Reverse Transcription of cDNA followed by Polymerase Chain Reaction (PCR) and quantitative real-time PCR (qPCR) technique. Herein, The RNA target from the organ from VNN-positive grouper fish samples targeted RNA2 region with 230-bp in electrophoresis gel and 69-bp by TAM-probe from qPCR were successfully converted and amplified from all RNA extraction kits. Different results of VNN amplified-cDNA were RNA extraction methods-dependent. Amplification using Tri Reagent® extraction kit showed the best result, with a large amount of RNA genome with good purity and showed clear results from PCR and qPCR. Furthermore, the sensitivity of RNA extraction methods was different from 50-100 ng total RNA/reaction. It was concluded that all RNA extraction kits can be used for the detection of the VNN genome by PCR and qPCR technique while resulting in different RNA yields. Overall, this study offers suitable methods to extract the VNN genome from grouper fish.&#x0D;