Characterizing the viral genome and host response
Respiratory infections are prevalent around the world and impose an enormous health and economic burden. It is essential to understand the nature of the pathogen and diagnose accordingly as soon as possible to prevent its rapid spread and minimize mortality rates. With the advent of molecular biology techniques like genomics, information on these pathogens' identity can be obtained at scale by whole-genome sequencing. This approach includes purifying the pathogen from infected patient samples such as bronchoalveolar fluids, nasal or throat swab, or saliva, and sequencing the pathogen's genome. However, this method poses technical challenges, as pathogen genome content can be scarce and/or degraded. In the case of RNA viruses, researchers need RNA-seq methods that are sensitive and can deal with potentially degraded viral genomic material.
Since the COVID-19 outbreak, researchers worldwide have generated genomics data to define the viral genome and track mutations precisely. The widespread analysis of SARS-CoV-2 genomes, as the epidemic was expanding into a pandemic, allowed researchers to characterize the evolution of the virus. This information is used to deepen our understanding of infection routes and viral sequence variability that might be contributing to the clinical course of the disease. Whole-genome sequencing of SARS-CoV-2 is critical for vaccine design as it provides valuable information on potential antigenic epitopes or new viral variants that could trigger or impair an immune response. This approach is used in reverse vaccinology, which aims to identify promising vaccine candidates through bioinformatics analysis of the viral genome (Bidmos et al. 2018). Our RNA-seq kit SMARTer Stranded total RNA-seq kit v2 (Pico v2; Figure 1) was used for SARS-CoV-2 sequencing in the early stages of the outbreak to provide full-length sequences of the virus (Wu et al. 2019). Examples of published work utilizing SMARTer Stranded RNA-seq technology within workflows for studying and developing vaccines against various pathogens, including coronaviruses, are summarized in a table at the end of this page. We recently launched Pico v3, an improved version of the Pico v2 kit.
Figure 1. Transcript expression levels for a 10-ng input amount is shown for the Pico v2 kit. Panel A. Higher reproducibility was observed between 1-ng and 10-ng inputs for data generated with the Pico v2 kit. FPKM values are shown on a log10 scale. Transcripts represented in only one library can be seen along the X- and Y-axes of the scatter plots. Panel B. A summary of the features and benefits of the Pico v2 kit.
In addition to focusing on the viral genome itself, researchers also study the variable regions of T-cell- and B-cell-receptor repertoires of infected patients, providing useful information for identifying novel protective antigens and assess vaccine candidate's efficacy. Studying the immune system of infected patients poses challenges similar to viral genome analysis, as infectious conditions often lead to lymphopenia in patients suffering from viral infections.
Our SMARTer immune profiling kits are used by a number of research groups to assess TCR and BCR repertoire diversity in various disease conditions (see citations below). The features and benefits of the SMARTer TCR and BCR immune profiling kits are summarized below.
Figure 2. Determining sequencing saturation point with the SMARTer human BCR kit's UMI-based error correction gives you more confidence in your clonotype calls and reduces sequencing cost. Panel A. BCR profiling libraries from 10 ng of single-donor PBMC RNA were prepared for this experiment using the SMARTer Human BCR IgG IgM H/K/L Kit. Sequencing saturation for SMARTer human BCR library and IgG clonotype counts at different sequencing depths with (blue line) and without (purple line) UMI-based error correction is shown. With UMIs, you can determine the sequencing saturation point so you don't have to sequence more than needed. All analyses at different sequencing depths were generated with our Takara Bio Immune Profiler Software. Panel B. A summary of the features and benefits of our immune profiling kits.
Citations for products used for viral and host genome sequencing and immune profiling
| Title | Link | Product |
|---|---|---|
| SMARTer Stranded total RNA-seq kit (Pico v2) |
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| SARS-CoV-2 | ||
| SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients |  |
SMARTer Stranded total RNA-seq kit v2 |
| Genomic Epidemiology of SARS-CoV-2 in Guangdong Province, China | |
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| Complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in Wuhan, China | |
|
| Complete Genome Sequence of a SARS-CoV-2 Strain Isolated in Northern Germany | |
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| Genomics functional analysis and drug screening of SARS-CoV-2 | |
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| Coding-Complete Genome Sequences of Two SARS-CoV-2 Isolates from Early Manifestations of COVID-19 in Israel | |
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| Shared SARS-CoV-2 diversity suggests localised transmission of minority variants | |
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| Other viruses | ||
| A novel Polycipiviridae virus identified in Pteropus lylei stools | |
SMARTer Stranded total RNA-seq kit v2 |
| High resolution metagenomic characterization of complex infectomes in paediatric acute respiratory infection | |
|
| Novel human reovirus isolated from children and its long-term circulation with reassortments | |
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| Simultaneous Viral Whole-Genome Sequencing and Differential Expression Profiling in Respiratory Syncytial Virus | |
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| SMARTer human TCR/BCR profiling kits | ||
| Co-activation of macrophages and T cells contribute to chronic GVHD in human IL-6 transgenic humanised mouse model | |
SMARTer Human TCR a/b Profiling Kit |
| Identification of a neoantigen epitope in a melanoma patient with good response to anti-PD-1 antibody therapy | |
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| Clinically relevant cytotoxic immune cell signatures and clonal expansion of T-cell receptors in high-risk MYCN-not-amplified human neuroblastoma | |
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| Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells | |
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| Recovery and assessment of leukocytes from LR Express filters | |
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| Immunological ignorance is an enabling feature of the oligo-clonal T cell response to melanoma neoantigens | |
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| Genomic profiling of intestinal T-cell receptor repertoires in inflammatory bowel disease | |
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