Metatranscriptomics to characterize respiratory virome, microbiome, and host response directly from clinical samples

Abstract

We developed a metatranscriptomics method that can simultaneously capture the respiratory virome, microbiome, and host response directly from low-biomass clinical samples. Using nasal swab samples, we have demonstrated that this method captures the comprehensive RNA virome with sufficient sequencing depth required to assemble complete genomes. We find a surprisingly high-frequency of Respiratory Syncytial Virus (RSV) and Coronavirus (CoV) in healthy children, and a high frequency of RSV-A and RSV-B co-infections in children with symptomatic RSV. In addition, we have characterized commensal and pathogenic bacteria, and fungi at the species-level. Functional analysis of bacterial transcripts revealed H. influenzae was highly active in symptomatic RSV subjects. Host transcriptome shows upregulation of the innate immune system, antiviral response and inflammasome pathway, and down regulation of fatty-acids pathway. This method is broadly applicable to infer transcriptome landscape of an infected system, surveil respiratory infections, and to sequence RNA viruses directly from clinical samples.

Mucosal gene expression in response to SARS-CoV-2 is associated with early viral load

“…Total RNA from the nasopharyngeal swab samples was extracted and further processed as previously described [10]. After quality-based trimming and removal of human and bacterial rRNA reads, we had an average of 98,120,791 (740,58,180 -median) human reads and an average of 2,263,290 (848,478 -median) microbiome reads.…”

“…The nasal swab samples in the self-collection kit (FLOQSwabs, Copan Diagnostics Inc.) were vortexed for 2 minutes, then an aliquot of 250µL nasal swap sample was used for RNA extraction. The total RNA from these samples were extracted as describe previously [10]. In brief, 250µL aliquot from the nasal swab sample was homogenized in 600µL QIAzol (Qiagen) and 500µL of 2.0 mm zirconium oxide beads (Next Advance, Inc. Cat: ZROB05) using a Bullet Blender homogenizer (BB24-AU, Next Advance, Inc).…”

“…However, little is known about the relationship between SARS-CoV-2 viral load and airway mucosal immune response. To fill this gap in knowledge, we profiled the host mucosal transcriptome from nasopharyngeal samples collected from outpatient adults infected with SARS-CoV-2 during Spring 2020 with mild-to-moderate symptoms utilizing a comprehensive metatranscriptomic method we recently developed [10]. We examined the association of SARS-CoV-2 viral load with the mucosal immune response.…”

A Universal Day Zero Infectious Disease Testing Strategy Leveraging CRISPR-based Sample Depletion and Metagenomic Sequencing

“…NGS can fulfill those requirements. Shotgun NGS generates sequence information from every molecule in a sample (e.g., nasal swab, saliva, blood), and has shown to be applicable for human infectious disease diagnostic tests (7)(8)(9), respiratory infections (10), and universal pathogen detection (11)(12)(13)(14)(15). Prior to the pandemic, an NGS-based study involving a population of ~1,000 pangolins from a wet market in Wuhan China, identified coronavirus infections in over 70% of the pangolin samples tested (16).…”