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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.

SPLIT™ RNA Extraction Kit

Lexogen’s RNA Extraction Kit SPLIT is a universal kit to isolate RNA from diverse sources such as tissue, cells and body fluids. It has already been shown to be highly effective in the extraction of RNA viruses such as HIV, HCV, and HEV from human plasma (Manso et al., 2017). Learn more about SPLIT RNA Extraction Kit.

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 efficiently removes ribosomal RNA from intact and degraded total RNA samples and makes low abundant viral RNA accessible for analysis by RNA sequencing (RNA-Seq). 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.

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™-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.

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.

Further Products for Virus Research: SLAMseq and SIRVs

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

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

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

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

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