scholarly journals Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

2021 ◽  
Author(s):  
Bingyu Yan ◽  
Srishti Chakravorty ◽  
Carmen Mirabelli ◽  
Luopin Wang ◽  
Jorge L. Trujillo-Ochoa ◽  
...  
Keyword(s):  
Life Cycle ◽  
Reverse Transcriptase ◽  
Human Genome ◽  
Rna Sequencing ◽  
Rna Virus ◽  
Template Switching ◽  
Rna Seq ◽  
Positive Sense ◽  
Infected Cells ◽  
Nuclear Phase

Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive-sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle. Thus, it is biologically unlikely to be in a location where splicing events could result in genome integration. Therefore, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ∼1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background “noise”. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes. Importance The pathogenic mechanisms underlying SARS-CoV2, the virus responsible for COVID-19, are not fully understood. In particular, relatively little is known why some individuals develop life-threatening or persistent COVID-19. Recent studies identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV2 infected cells and suggested that HVC events support potential “human genome invasion” and “integration” by SARS-CoV2. This suggestion has fueled concerns about the long-term effects of current mRNA vaccines that incorporate elements of the viral genome. SARS-CoV2 is a positive-sense, single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events. Here we provide several evidences suggesting that the observed HVC events are likely artifactual.

scholarly journals SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA

2021 ◽  
Vol 12 ◽  
Author(s):  
Anastasiya Kazachenka ◽  
George Kassiotis
Keyword(s):  
Rna Sequencing ◽  
Nuclear Dna ◽  
Rna Virus ◽  
Rna Seq ◽  
Dna Viruses ◽  
Intergenic Sequences ◽  
Infected Cells ◽  
Chimeric Rna ◽  
Virus Integration ◽  
Host Genes

The human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, based on a number of observations. One key observation was the presence of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin specifically of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA were highly enriched for host exonic, rather than intronic or intergenic sequences and often involved the same, highly expressed host genes. Although these findings do not rule out SARS-CoV-2 somatic integration, they nevertheless suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.

scholarly journals SARS-CoV-2-host chimeric RNA-sequencing reads do not necessarily signify virus integration into the host DNA

2021 ◽  
Author(s):  
Anastasiya Kazachenka ◽  
George Kassiotis
Keyword(s):  
Rna Sequencing ◽  
Nuclear Dna ◽  
Rna Virus ◽  
Rna Seq ◽  
Dna Viruses ◽  
Intergenic Sequences ◽  
Infected Cells ◽  
Chimeric Rna ◽  
Virus Integration ◽  
Host Genes

ABSTRACTThe human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, partly based on the detection of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA was highly enriched for host exonic, than intronic or intergenic sequences and often involved the same, highly expressed host genes. These findings suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.

scholarly journals Optimization of library preparation based on SMART for ultralow RNA-seq in mice brain tissues

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Erteng Jia ◽  
Huajuan Shi ◽  
Ying Wang ◽  
Ying Zhou ◽  
Zhiyu Liu ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Single Cell ◽  
Rna Sequencing ◽  
Rna Structure ◽  
Treatment Plan ◽  
Template Switching ◽  
Rna Seq ◽  
Gene Detection ◽  
Experimental Conditions ◽  
Optimized Protocol

Abstract Background Single-cell RNA sequencing (scRNA-seq) provides new insights to address biological and medical questions, and it will benefit more from the ultralow input RNA or subcellular sequencing. Results Here, we present a highly sensitive library construction protocol for ultralow input RNA sequencing (ulRNA-seq). We systematically evaluate experimental conditions of this protocol, such as reverse transcriptase, template-switching oligos (TSO), and template RNA structure. It was found that Maxima H Minus reverse transcriptase and rN modified TSO, as well as all RNA templates capped with m7G improved the sequencing sensitivity and low abundance gene detection ability. RNA-seq libraries were successfully prepared from total RNA samples as low as 0.5 pg, and more than 2000 genes have been identified. Conclusions The ability of low abundance gene detection and sensitivity were largely enhanced with this optimized protocol. It was also confirmed in single-cell sequencing, that more genes and cell markers were identified compared to conventional sequencing method. We expect that ulRNA-seq will sequence and transcriptome characterization for the subcellular of disease tissue, to find the corresponding treatment plan.

scholarly journals Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

2021 ◽  
Author(s):  
Bingyu Yan ◽  
Srishti Chakravorty ◽  
Carmen Mirabelli ◽  
Luopin Wang ◽  
Jorge L. Trujillo-Ochoa ◽  
...  
Keyword(s):  
Genome Rearrangement ◽  
High Throughput Sequencing ◽  
Rna Virus ◽  
Fruit Fly ◽  
Template Switching ◽  
Rna Seq ◽  
Sequencing Technologies ◽  
Cellular Genes ◽  
Infected Cells ◽  
Alignment Errors

AbstractPathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background “noise”. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.

scholarly journals A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

2015 ◽  
Vol 90 (3) ◽  
pp. 1278-1289 ◽  
Author(s):  
Catrin Stutika ◽  
Andreas Gogol-Döring ◽  
Laura Botschen ◽  
Mario Mietzsch ◽  
Stefan Weger ◽  
...  
Keyword(s):  
Life Cycle ◽  
Fusion Protein ◽  
Rna Sequencing ◽  
Splice Variants ◽  
Productive Infection ◽  
Minus Strand ◽  
Rna Seq ◽  
Adeno Associated Virus ◽  
Divergent Transcription ◽  
Productive Replication

ABSTRACTAdeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5ˈ rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad.IMPORTANCENext-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAVin vivo, combinedin cellulaeandin silicostudies will help to forward the understanding of the unique, bipartite AAV life cycle.

scholarly journals Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

2021 ◽  
Author(s):  
Yu-Sheng Chen ◽  
Shuaiyao Lu ◽  
Bing Zhang ◽  
Tingfu Du ◽  
Wen-Jie Li ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Comprehensive Analysis ◽  
Host Genome ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Rna Seq ◽  
Sequencing Data ◽  
Infected Cells

SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didn't identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2's integration into host genome is lacking.

scholarly journals Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

2022 ◽  
Vol 5 (4) ◽  
pp. e202101124
Author(s):  
Elena Rensen ◽  
Stefano Pietropaoli ◽  
Florian Mueller ◽  
Christian Weber ◽  
Sylvie Souquere ◽  
...  
Keyword(s):  
Human Tissue ◽  
Rna Virus ◽  
Source Code ◽  
Initial Application ◽  
Green Monkey ◽  
Positive Sense ◽  
Therapeutic Molecules ◽  
Infected Cells ◽  
Detailed Protocol ◽  
Transcription And Replication

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

scholarly journals Slinker: Visualising novel splicing events in RNA-Seq data

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 1255
Author(s):  
Breon Schmidt ◽  
Marek Cmero ◽  
Paul Ekert ◽  
Nadia Davidson ◽  
Alicia Oshlack
Keyword(s):  
Rare Disease ◽  
Human Genome ◽  
Rna Sequencing ◽  
Reference Genome ◽  
Data Driven ◽  
Rna Seq ◽  
Bioinformatics Pipeline ◽  
Link Type ◽  
Muscular Disorders ◽  
Data Driven Approach

Visualisation of the transcriptome relative to a reference genome is fraught with sparsity. This is due to RNA sequencing (RNA-Seq) reads being predominantly mapped to exons that account for just under 3% of the human genome. Recently, we have used exon-only references, superTranscripts, to improve visualisation of aligned RNA-Seq data through the omission of supposedly unexpressed regions such as introns. However, variation within these regions can lead to novel splicing events that may drive a pathogenic phenotype. In these cases, the loss of information in only retaining annotated exons presents significant drawbacks. Here we present Slinker, a bioinformatics pipeline written in Python and Bpipe that uses a data-driven approach to assemble sample-specific superTranscripts. At its core, Slinker uses Stringtie2 to assemble transcripts with any sequence across any gene. This assembly is merged with reference transcripts, converted to a superTranscript, of which rich visualisations are made through Plotly with associated annotation and coverage information. Slinker was validated on five novel splicing events of rare disease samples from a cohort of primary muscular disorders. In addition, Slinker was shown to be effective in visualising deletion events within transcriptomes of tumour samples in the important leukemia gene, IKZF1. Slinker offers a succinct visualisation of RNA-Seq alignments across typically sparse regions and is freely available on Github.

The Mauriceville and Varkud plasmids: primitive retroelements found in Neurospora mitochondria

1995 ◽  
Vol 73 (S1) ◽  
pp. 173-179 ◽  
Author(s):  
Alan M. Lambowitz ◽  
Chia-Chien Chiang
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
De Novo ◽  
Rna Virus ◽  
Life Forms ◽  
Template Switching ◽  
Cdna Synthesis ◽  
Evolutionary Connection ◽  
Circular Dnas ◽  
Rna And Dna

The Mauriceville and closely related Varkud plasmids are small circular DNAs (3.6 and 3.7 kb, respectively) found in the mitochondria of certain Neurospora spp. strains isolated from nature. The plasmids replicate via reverse transcription and appear to be primitive retroelements that may be related to the early ancestors of retroviruses. Recent studies have shown that the plasmid reverse transcriptase closely resembles certain viral RNA-dependent RNA polymerases in initiating (−) strand cDNA synthesis de novo (i.e., without a primer) at a tRNA-like structure at the 3′ end of the plasmid transcript. The plasmid reverse transcriptase can also use DNA or RNA primers and can carry out template-switching reactions that lead to the generation of suppressive mutant plasmids or the integration of the plasmids into mitochondrial DNA. The characteristics of the plasmids and their reverse transcription mechanism suggest an evolutionary connection between RNA and DNA replication and raise the possibility that the plasmids are related to the earliest DNA-based life forms that emerged at the time of transition from an RNA to a DNA world. Key words: DNA synthesis, evolution, retrovirus, reverse transcriptase, RNA virus.

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