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The new coronavirus variant

The recent global outbreak of the novel coronavirus, SARS-CoV-2, has researchers focusing their attention on understanding this devastating pathogen. The coronaviruses (CoVs) are capable of causing mild illnesses such as a common cold to devastating diseases such as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) (Wit et al., 2016). The novel CoV variant, SARS-CoV-2, was identified in humans late December 2019 and has since rapidly spread throughout society, recently being classified as a pandemic (WHO, 2020). Research has therefore intensified to understand the molecular biology and genetics of SARS-CoV-2 to develop improved diagnostic and therapeutic tools. SARS-CoV-2 has a positive-strand RNA genome of about 30 kb length that is transcribed into 10 subgenomic RNAs, adding complexity to this viral transcriptome (Taiaroa et al., 2020).

Enabling complete transcriptome analysis

Lexogen, a transcriptomics company based in Vienna, Austria and New Hampshire, USA, provides a plethora of technologies, products, and services that are ideally positioned for SARS-CoV-2 research. Below we have described products that will specifically accelerate and empower your viral research, taking you from RNA extraction to ribosomal RNA depletion, total and targeted RNA-Seq library preparation (with optimized indexing), and NGS data evaluation.

Need a robust method for extraction of the viral RNA?

SPLIT™ Rapid Viral RNA/DNA Extraction Kit

The SPLIT Rapid Viral RNA/DNA Extraction Kit enables the isolation of high-quality RNA & DNA from liquid samples using a fast, streamlined protocol. This column-based protocol has been validated externally for extraction of SARS-CoV-2 RNA with buccal swab samples in virus transport medium (VTM).Learn more about SPLIT Rapid Viral RNA/DNA Extraction Kit.

SARS-CoV-2 Rapid RNA Extraction Kit

The SARS-CoV-2 Rapid RNA Extraction Kit provides a streamlined protocol for isolation of nucleic acids from liquid samples such as nasopharyngeal and buccal swabs or gargle samples within 15 minutes and can be automated on liquid handlers. It is a quick and easy bead-based purification and can be directly used for molecular biology applications such as qRT-PCR, reverse transcription, and targeted RNA Sequencing. Learn more about SARS-CoV-2 Rapid RNA Extraction Kit.

Analyzing host RNA and want to deplete ribosomal RNA or select for poly(A) RNA?

RIBOCOP™ rRNA Depletion Kit and Poly(A) RNA Selection Kit

The majority (80-90%) of RNA extracted from human samples consists of ribosomal RNA that is not of interest and consumes valuable sequencing reads. Lexogen’s RiboCop rRNA depletion kit removes ribosomal RNA from intact and degraded total RNA samples and makes low abundant viral RNA accessible for analysis by RNA sequencing (RNA-Seq). RiboCop has already been used to efficiently deplete ribosomal RNA from SARS-infected cells (Mulay et al., 2020; Wyler et al., 2020): Learn more about RiboCop rRNA Depletion Kit.

Alternatively, the Poly(A) RNA Selection Kit offers the opportunity to select for 3’ polyadenylated RNAs (such as cellular mRNAs and coronavirus transcripts). Learn more about Poly(A) RNA Selection Kit.

Need a robust library preparation protocol for virus and host RNA-Seq?

CORALL™ Total RNA-Seq Library Preparation and Automated Data Evaluation on the BlueBee® platform

CORALL is a universal NGS library preparation kit for analysis of total RNA, rRNA-depleted RNA and poly(A)-selected RNA on Illumina short-read platforms. Its high sensitivity (minimum input of only 1 ng rRNA-depleted RNA) and superior 5’-end to 3’-end coverage makes it an ideal choice to research RNA virus genomes full-length. Novel, 12 nucleotide long Unique Dual Barcode Indices (UDIs) are available for CORALL for multiplexing of up to 384 libraries with perfect inter-index distance and optimal read recovery, maximizing the sequencing output. Learn more about CORALL Total RNA-Seq Library Prep Kit.

Data analysis can be performed automated on the BlueBee Genome Analysis Platform, with CORALL data analysis pipelines installed and validated from quality control and mapping of the reads to gene expression quantification. In addition to more than 40 pre-installed genomes, the SARS-CoV-2 genome (NCBI Reference Sequence NC_045512.2) is now available as well to facilitate coronavirus research. Learn more about Automated Data Evaluation on the BlueBee® platform.

QUANTSEQ™ 3′ mRNA-Seq Library Prep Kit

The QuantSeq 3’ mRNA-Seq Kit provides a library preparation protocol designed to generate Illumina compatible NGS libraries of sequences close to the 3’ end of poly(A) RNA. Only one fragment per transcript is generated, thereby enabling accurate gene expression quantification while saving sequencing depth. Available indexing solutions include UDIs and allow multiplexed sequencing of up to 9,216 samples.

QuantSeq has already been used to assess gene expression changes in cellular and organoid systems employed for SARS-CoV-2 research (Giobbe et al., 2020; Samuel et al., 2020; Tian et al., 2021; Verstockt et al., 2020).

QUANTSEQ™-FLEX Targeted RNA-Seq Library Preparation Kit

In its flexible version, Lexogen is offering the benefits of the highly efficient and successful QuantSeq 3’ mRNA-Seq kit for targeted sequencing projects. Gene expression panels can be designed that are (A) amplifying a set of amplicons using specific first strand and specific second strand primers or (B) are 3’ poly(A)-specific using the standard QuantSeq first strand oligo(dT) primer and targeted second strand primers. QuantSeq-Flex users can design their own primers to detect viral RNA, e.g. based on sequences that were already chosen for diagnostic RT-PCR assays (Corman et al., 2020) or published virus sequences to cover regions of interest (also by designing overlapping amplicons). In all cases, QuantSeq-Flex offers a tested kit design for deep-sequencing parts of the transcriptome with low read-depth. It is based on QuantSeq advantages such as highest sensitivity & reproducibility and a highly competitive cost structure owned to an effective workflow, low kit costs and a maximum of multiplexing capacity. Up to 9,216 6-nt dual indexing combinations are available for QuantSeq-Flex, as well as Lexogen’s new 12-nt UDI system with a pre-designed and optimized set of 384 barcodes. Learn more about QuantSeq-Flex Targeted RNA-Seq Library Prep Kit.

Further Products for Virus Research: TeloPrime, SLAMseq, SIRVs

TELOPRIME™ Full-Length cDNA Synthesis and Amplification Kit

TeloPrime is a unique kit, providing full-length cDNA synthesis of RNAs that are both polyadenylated and capped. The amplified cDNAs can then be sequenced using long-read technologies from Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). Virus research has benefited widely from this combination, with the viral RNA genomes of PRV (Moldován et al., 2017), HCMV (Balázs et al., 2018), AcMNPV (Boldogkői et al., 2018), VZV (Prazsák et al., 2018), and HCV (Tombácz et al., 2019) already sequenced using TeloPrime and the ONT platform.

The SARS-CoV-2 genome and its subgenomic RNAs are the longest ones of all RNA viruses, and hence, obtaining a full-length representation is difficult. However, since the SARS-CoV-2 transcripts have been shown to be both polyadenylated (Taiaroa et al., 2020) and likely capped (Chen et al., 2016), they are accessible for TeloPrime full-length cDNA synthesis. Downstream analysis can benefit greatly from focusing on intact transcripts that are characterized by having both terminal modifications, without sequencing degraded or incomplete viral transcripts. Learn more about TeloPrime Full-Length cDNA Amplification Kit.

Lexogen is also the provider of SLAMseq, a metabolic labeling method that among its manifold applications has already been shown to be able to unambiguously differentiate cell infection by viruses from carryover of viral nucleic acids (Cheval et al., 2019). Further, the company provides external RNA-Seq control transcripts; these Spike-In RNA Variants (SIRVs) are designed to validate any RNA sequencing workflow, and among the modules offered are industry-leading long transcripts with up to 12 kb length. These are currently available for early access users and particularly useful for the evaluation of long-read platforms that are capable to sequence coronavirus RNAs full-length.

Lexogen is focusing exclusively on offering kits and services for transcriptome analysis and has built a sophisticated and extensive product portfolio that can be accessed at www.lexogen.com. For further information and ordering please e-mail info@lexogen.com or contact us at +43-1-3451212-41 (all countries except US) and +1-603-431-4300 (US only).

References

Balázs, Zsolt; Tombácz, Dóra; Szűcs, Attila; Snyder, Michael; Boldogkői, Zsolt (2018): Dual Platform Long-Read RNA-Sequencing Dataset of the Human Cytomegalovirus Lytic Transcriptome. Frontiers in Genetics 9, S. 432. DOI: 10.3389/fgene.2018.00432

Boldogkői, Zsolt; Moldován, Norbert; Szűcs, Attila; Tombácz, Dóra (2018): Transcriptome-wide analysis of a baculovirus using nanopore sequencing. Scientific data 5, S. 180276. DOI: 10.1038/sdata.2018.276

Chen, Yu; Su, Ceyang; Ke, Min; Jin, Xu; Xu, Lirong; Zhang, Zhou et al. (2011): Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2′-O-methylation by nsp16/nsp10 protein complex. PLoS Pathogens 7 (10), e1002294. DOI: 10.1371/journal.ppat.1002294

Cheval, Justine; Muth, Erika; Gonzalez, Gaëlle; Coulpier, Muriel; Beurdeley, Pascale; Cruveiller, Stéphane; Eloit, Marc (2019): Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids. mSphere 4 (3). DOI: 10.1128/mSphere.00298-19

Corman, Victor M.; Landt, Olfert; Kaiser, Marco; Molenkamp, Richard; Meijer, Adam; Chu, Daniel K. W. et al. (2020): Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 25 (3). DOI: 10.2807/1560-7917.ES.2020.25.3.2000045

Giobbe, Giovanni, G.; Bonfante, Francesco; Zambaiti, Elisa; Gagliano, Onelia; Jones, Brendan C.; Luni, Camilla et al. (2020): SARS-CoV-2 infection and replication in human fetal and pediatric gastric organoids. BioRxiv. doi: 2020.06.24.167049

Manso, Carmen F.; Bibby, David F.; Mbisa, Jean L. (2017): Efficient and unbiased metagenomic recovery of RNA virus genomes from human plasma samples. Scientific Reports 7 (1), p. 4173. DOI: 10.1038/s41598-017-02239-5

Moldován, Norbert; Tombácz, Dóra; Szűcs, Attila; Csabai, Zsolt; Snyder, Michael; Boldogkői, Zsolt (2017): Multi-Platform Sequencing Approach Reveals a Novel Transcriptome Profile in Pseudorabies Virus. Frontiers in Microbiology 8, S. 2708. DOI: 10.3389/fmicb.2017.02708

Mulay, A.; Konda, B.; Garcia, G. Jr; Yao, C.; Beil, B.; Sen, C. et al. (2020): SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery. BioRxiv. DOI: 10.1101/2020.06.29.174623

Prazsák, István; Moldován, Norbert; Balázs, Zsolt; Tombácz, Dóra; Megyeri, Klára; Szűcs, Attila et al. (2018): Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus. BMC Genomics 19 (1), S. 873. DOI: 10.1186/s12864-018-5267-8

Samuel, Ryan M.; Majd, H.; Richter, Mikayla N.; Ghazizadeh, Zaniar; Maryam Zekavat, Seyedeh; Navickas, Albertas et al. (2020): Androgen Signaling Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men. Cell Stem Cell 27 (6) p. 876–889.e12. doi: 10.1016/j.stem.2020.11.009

Taiaroa, George; Rawlinson, Daniel; Featherstone, Leo; Pitt, Miranda; Caly, Leon; Druce, Julian et al. (2020): Direct RNA sequencing and early evolution of SARS-CoV-2. bioRxiv 34. DOI: 10.1101/2020.03.05.976167

Tian, Ruilin; Samelson, Avi J.; Rezelj, Veronica V.; Chen, Merissa; Ramadoss, Gokul N.; Guo, Xiaoyan et al. (2021): BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2. BioRxiv. doi: 10.1101/2021.01.19.427194

Tombácz, Dóra; Moldován, Norbert; Balázs, Zsolt; Gulyás, Gábor; Csabai, Zsolt; Boldogkői, Miklós et al. (2019): Multiple Long-Read Sequencing Survey of Herpes Simplex Virus Dynamic Transcriptome. Front. Genet. 10, S. 15989. DOI: 10.3389/fgene.2019.00834

Wit, Emmie de; van Doremalen, Neeltje; Falzarano, Darryl; Munster, Vincent J. (2016): SARS and MERS: recent insights into emerging coronaviruses. Nature Reviews Microbiology 14 (8), p. 523–534. DOI: 10.1038/nrmicro.2016.81

World Health Organization (2020). Statement on the second meeting of the International Health Regulations (2005) Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV). 30 January 2020

Verstockt, Bram; Verstockt, Sare; Rahiman, Saeed A.; Ke, Bo-jun; Arnauts, Kaline; Cleynen, Isabelle et al. (2020): Intestinal Receptor of SARS-CoV-2 in Inflamed IBD Tissue Seems Downregulated by HNF4A in Ileum and Upregulated by Interferon Regulating Factors in Colon. Journal of Crohn’s and Colitis, jjaa185, DOI: 10.1093/ecco-jcc/jjaa185

Wyler, Emanuel; Mösbauer, Kirstin; Franke, Vedran; Diag, Asija; Gottula, Lina T.; Arsie, Roberto et al. (2020): Bulk and single-cell gene expression profiling of SARS-CoV-2 infected human cell lines identifies molecular targets for therapeutic intervention. BioRxiv. DOI: 10.1101/2020.05.05.079194