zr viral rna kit  (Zymo Research)

Journal: bioRxiv

Article Title: Unique nasal cell states induced by common pediatric respiratory viruses

doi: 10.64898/2026.04.20.719671

Figure Lengend Snippet: A. Schematic of participant cohort and study design. RSV = respiratory syncytial virus; scRNA-seq = single-cell RNA sequencing. Participant metadata is summarized in Supplementary Table 1 and provided in full in Supplementary Table 2 . B. Heatmap of participant cohort colored by assigned viral group (top), the presence of acute respiratory symptoms (middle), and viral RNA cycle threshold (CT) value from supernatant collected during swab processing measured by qPCR (bottom). 1 asymptomatic participant was assigned based on a positive PCR from clinically sampled viral transport media. Not detected: CT value >40 or undetectable; Not measured: supernatant was not available; Rhino = rhinovirus. C. Age of each participant in months by viral group. SCoV2 = SARS-CoV-2. D. Distribution of sex within each viral group. E. Distribution of reported duration of illness within each viral group. *chi-sq. standardized Pearson residual > 2. F. Distribution of hospital admission within each viral group. Control samples are restricted to those collected at Boston Children’s Hospital (BCH). ICU = intensive care unit. G. Distribution of wheezing groups (see Methods for assignment criteria) within each viral group. Control samples are restricted to those collected at BCH. Tx = wheezing-directed medication. H. UMAP of all cells (n=335,174) from all participants colored by cell type. Cell type labels were assigned after iterative clustering. cDCs = conventional dendritic cells; pDCs = plasmacytoid dendritic cells; LTi = lymphoid tissue inducer cells; NK = natural killer. I. Hierarchical tree of all 79 cell subsets identified after iterative clustering. End nodes are colored by cell type of origin. Hierarchical distances were calculated from pseudobulk aggregates of each cell subset. See for subset labels. J. Frequency of specified cell subsets as a percentage of either all epithelial or all immune cells per sample, compared across viral groups. Samples with <100 epithelial or immune cells were excluded. Ctrl = Control. C,J : Boxplots represent the median (center line), interquartile range (box), and 1.5x the interquartile range (whiskers). Statistical test is Kruskal-Wallis test with Benjamini-Hochberg correction for multiple comparisons. Dunn’s post-hoc test, *p<0.05, **p<0.001, ***p<0.0001.

Article Snippet: RNA was extracted from 200 μL of the specimen using Quick RNA Viral kit (Zymo Research) following the manufacturer’s protocol. qPCR for RSV detection and subgrouping was performed using 6.25 μL RNA with Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) and previously published RSV-specific primers and probes .

Techniques: Virus, Single Cell, RNA Sequencing, Control

Journal: bioRxiv

Article Title: Unique nasal cell states induced by common pediatric respiratory viruses

doi: 10.64898/2026.04.20.719671

Figure Lengend Snippet: Related to A. Schematic for identifying viral RNA+ cells by alignment or Kraken2 metagenomics approach. See Methods for details. B. Violin plot demonstrating viral UMI/cell for epithelial compared to immune viral RNA+ cells for SARS-CoV-2, rhinovirus, and RSV. Rhino = rhinovirus. RSV = respiratory syncytial virus. C. Spearman correlation between viral RNA+ cells as a percentage of epithelial cells and inverse CT value from qRT-PCR of supernatant collected during swab processing and disease severity (see Methods for details) for each virus (top) and modified BROSJOD disease severity score (see Methods for details, bottom). SCoV2 = SARS-CoV-2. Spearman correlation test with Benjamini-Hochberg correction for multiple comparisons. *p<0.05, ***p<0.001. D. Heatmap of SARS-CoV-2 (left) and RSV (right) gene expression in viral RNA+ cells. Top bar depicts the individual participant for each cell. UMIs shown are post removal of background reads by Cellbender. E. Spearman correlation between viral RNA+ cells as a percentage of epithelial cells between alignment and Kraken methods for respective viral groups. R 2 and Spearman p values are shown. For RSV, two different Kraken2 levels of classification are depicted. The plot labeled “RSV” indicates the custom kraken database species-level identifier “Respiratory Syncytial Virus” compared to RSV+ cells by alignment method. The plot labeled “Orthopneumovirus” indicates the custom kraken database genus-level identifier “Orthopneumovirus” compared to RSV+ cells by alignment method. F. Violin plot of kraken k-mers for parainfluenza or human metapneumovirus (HMPV) per participant for participants with >1 viral RNA+ cell for parainfluenza or HMPV. Participants are labeled by viral group. Co. = coinfection. G. Dotplot of viral RNA+ cells identified by Kraken method in all subsets for SARS-CoV-2, rhinovirus, and RSV. Dot size represents the percentage of cells in the subset that are viral RNA+. Color represents the average viral k-mers across all cells in the subset. Cell subsets with <5% viral RNA+ cells are collapsed. Cell subsets with fewer than 100 cells/group were excluded. H. Dotplot of viral RNA+ cells by alignment method in each immune subset for SARS-CoV-2, rhinovirus, and RSV. Dot size represents the percentage of cells in the subset that are viral RNA+. Color represents the average viral transcripts (UMI) across all cells in the subset. Cell subsets with <1% viral RNA+ cells are collapsed. Cell subsets with fewer than 100 cells/group were excluded. I. Dotplot of SARS-CoV-2 and RSV viral receptor and entry factors within each epithelial cell type. Includes all epithelial cells from all participants. J. Venn diagram comparing marker genes for NFKBIA+ATF3+ ciliated cells in this dataset, "hyperinfected" ciliated cells identified in a SARS-CoV-2 human challenge study and a PER2+EGR1+GDF15+ ciliated subset consistently enriched in viral RNA+ cells across SARS-CoV-2 variants .

Article Snippet: RNA was extracted from 200 μL of the specimen using Quick RNA Viral kit (Zymo Research) following the manufacturer’s protocol. qPCR for RSV detection and subgrouping was performed using 6.25 μL RNA with Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) and previously published RSV-specific primers and probes .

Techniques: Metagenomics, Virus, Quantitative RT-PCR, Modification, Gene Expression, Labeling, Marker

Journal: bioRxiv

Article Title: Unique nasal cell states induced by common pediatric respiratory viruses

doi: 10.64898/2026.04.20.719671

Figure Lengend Snippet: A. UMAP of epithelial cells (n=224,431) from participants in control and monoinfection groups colored by cell subset. Pro-inflam = pro-inflammatory; MT-high = mitochondrial-high; IFN-stim = interferon -stimulated. B. Split UMAP of epithelial cells from each viral group (Control: n=34,031; SARS-CoV-2: n=39,836; Rhinovirus: n=61,198; RSV: n=89,366) colored by viral RNA status. C. Dotplot of viral RNA+ cells in each epithelial subset for SARS-CoV-2, rhinovirus, and RSV. Dot size represents the percentage of cells in the subset that are viral RNA+. Color represents the average viral transcripts (UMI) across all cells in the subset. Epithelial subsets with >5% of cells viral RNA+ were included, other subsets were collapsed. SCoV2 = SARS-CoV-2. D. Frequency of specified viral RNA+ cell subsets as a percentage of epithelial cells per sample, compared across viral groups. Samples with <100 epithelial cells were excluded. Boxplots represent the median (center line), interquartile range (box), and 1.5x the interquartile range (whiskers). Statistical test is Kruskal-Wallis test with Benjamini-Hochberg correction for multiple comparisons. Dunn’s post-hoc test, *p<0.05, **p<0.001, ***p<0.0001. Ctrl = Control; Rhino = Rhinovirus. E. Violin plot of marker genes for select viral RNA+ epithelial subsets. F. Differentially expressed (DE) genes between RSV RNA+ cells and SARS-CoV-2 RNA+ cells within the CXCL11 +IFN-stim ciliated subset. Participants with at least 20 RNA+ CXCL11 +IFN-stim ciliated cells (SARS-CoV-2: n=4; RSV: n=6) were included in pseudobulk DE analysis. Wald significance test with Benjamini-Hochberg correction. Genes with adjusted p<0.05 are shown. Full list of DE genes is provided in Supplementary Table 4 . G. RNA velocity streams for all epithelial cells split by viral group for Control, SARS-CoV-2, and RSV groups, colored by cell type with NFKBIA+ATF3+ ciliated, CXCL11 +IFN-stim ciliated, and KRT17+S100A9+ ciliated subsets highlighted. H. Heat map of inferred transcription factor (TF) activity, z-score normalized between groups of cells, for select viral RNA+ cells, split by NFKBIA+ATF3+ ciliated, CXCL11+ IFN-stim ciliated, and KRT17+S100A9+ ciliated subsets, showing the top 10 transcription factors for each group of cells.

Article Snippet: RNA was extracted from 200 μL of the specimen using Quick RNA Viral kit (Zymo Research) following the manufacturer’s protocol. qPCR for RSV detection and subgrouping was performed using 6.25 μL RNA with Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) and previously published RSV-specific primers and probes .

Techniques: Control, Marker, Activity Assay

Journal: bioRxiv

Article Title: Unique nasal cell states induced by common pediatric respiratory viruses

doi: 10.64898/2026.04.20.719671

Figure Lengend Snippet: A. Dotplot of IFN gene expression by epithelial subset. Epithelial subsets with >1% of cells expressing any IFN gene were included, other subsets were collapsed. B. Differentially expressed (DE) genes between RSV RNA+ cells and RSV RNA- cells within the KRT17+S100A9+ ciliated subset. Participants with at least 20 KRT17+S100A9+ ciliated cells (n=26) were included in pseudobulk DE analysis. Wald significance test with Benjamini-Hochberg correction. Genes with adjusted p<0.05 are shown. Full list of DE genes is provided in Supplementary Table 4 . C. Dotplot of IFN gene expression by immune subset. Immune subsets with >2% of cells expressing any IFN gene were included, other subsets were collapsed. D. Frequency of IFN-hi plasmacytoid dendritic cells (pDCs) as a percentage of immune cells per sample, compared across viral groups. Ctrl = Control; SCoV2 = SARS-CoV-2; Rhino = Rhinovirus. E. Pseudobulk pathway activity score per participant for interferon-alpha (IFNα) response in epithelial cells, compared across viral groups. ULM = univariate linear model. Signature provided in Supplementary Table 5 . F. Spearman correlation between pseudobulk epithelial IFNα pathway activity score and select participant metadata variables within each viral group. CT value represents viral RNA PCR from scRNA-seq swab supernatants. G. Principal component analysis (PCA) of cellular composition for each sample. PCA was run on center-log-ratio (CLR) normalized cell subset abundances with percent of variance explained by each PC labeled on x and y axes. Ellipses for each viral group are centered at centroid and represent 80% confidence region. H. Cell subsets ranked by loadings for principal components 1 (left) and 2 (right). Top 10 positive and negatively ranked subsets are shown. Mac. = macrophage; Cil. = ciliated; Deut. = deuterosomal. I. Frequency of specified IFN-responsive epithelial and immune subsets as a percentage of immune or epithelial cells per sample, compared across viral groups. J. Transcription factor (TF) activity z-scores for select TFs across select IFN-responsive epithelial and immune subsets. K. Spearman correlation between CLR-normalized abundance of select cell subsets and inverse CT value from viral RNA PCR of scRNA-seq swab supernatants for each virus. D,I: Participants with <100 immune or epithelial cells were excluded. D,E,I: Boxplots represent the median (center line), interquartile range (box), and 1.5x the interquartile range (whiskers). Statistical test is Kruskal-Wallis test with Benjamini-Hochberg correction for multiple comparisons. Dunn’s post-hoc test, *p<0.05, **p<0.001, ***p<0.0001.

Article Snippet: RNA was extracted from 200 μL of the specimen using Quick RNA Viral kit (Zymo Research) following the manufacturer’s protocol. qPCR for RSV detection and subgrouping was performed using 6.25 μL RNA with Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) and previously published RSV-specific primers and probes .

Techniques: Gene Expression, Expressing, Control, Activity Assay, Labeling, Virus

Journal: bioRxiv

Article Title: Unique nasal cell states induced by common pediatric respiratory viruses

doi: 10.64898/2026.04.20.719671

Figure Lengend Snippet: A. Pearson correlation of CLR-normalized cell subset abundances among control, SARS-CoV-2, RSV, and rhinovirus participants. Both rows and columns were clustered using hierarchical clustering with Euclidean distance and complete linkage. P values from correlation test adjusted with Benjamini-Hochberg correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001. ED = emergency department. B. Viral RNA level (1/CT value) from PCR of scRNA-seq swab supernatants for each sample in the RSV group divided by hospital admission status. ICU = intensive care unit. C. Principal component analysis (PCA) of cellular composition for each sample in RSV participants colored by admission groups. The percent of variance explained by each PC is labeled on x and y axes. PCA was run on center-log-ratio (CLR) normalized cell subset abundances. See for PC loadings. D. Frequency of specified subsets as a percentage of epithelial or immune cells in RSV participants compared across admission groups. Disc. = Discharged, Admit. floor = Admitted to floor, Admit ICU = Admitted to ICU. E. Differentially expressed (DE) genes in all T cells between RSV participants admitted to ICU or admitted to the floor by pseudobulk differential expression. Wald significance test with Benjamini-Hochberg correction. Genes with adjusted p value of <0.05 are displayed. Full list of DE genes is provided in Supplementary Table 4 . F. Pseudobulk pathway activity score for IFNɑ (left) and IFNɣ (right) response genes per RSV participant in all cells, compared across admission groups. ULM = univariate linear model. Signatures provided in Supplementary Table 5 . G. PCA of cellular composition for RSV and Rhinovirus participants colored by wheezing group (see for group breakdown). Treatment included nebulized bronchodilator and/or corticosteroids. H. Machine-learning-derived subset importance for distinguishing no wheeze vs wheeze + treatment for Rhinovirus participants by Caret’s penalized generalized linear (GLMNet) model. Color of dot indicates the difference in center-log-ratio (CLR)-normalized cell subset abundance between patients with wheeze+tx compared to no wheeze. Dot size represents frequency of the cell subset, averaged across all included rhinovirus participants. All subsets that contributed to model importance are shown. Cil. = ciliated; Gob. = goblet; Deut. = deuterosomal; Iono. = ionocytes; Squam = squamous; Mac. = macrophages. I. Frequency of specified subsets as a percentage of epithelial or immune cells for RSV and Rhinovirus participants categorized as having no wheeze/no treatment or wheeze + treatment. Tx = treatment; Rhino = rhinovirus. J. Transcription factor (TF) network analysis for the top 3 transcription factors within the CST1 + goblet cell subset. Boxes: transcription factors colored by enrichment score, circles: target genes colored by gene expression relative to all epithelial cells. K. Violin plot of marker genes for CST1+ goblet compared to all other goblet cells. D,I: Participants with <100 epithelial or immune cells were excluded. B,D,F,I: Boxplots represent the median (center line), interquartile range (box), and 1.5x the interquartile range (whiskers). Statistical test is Kruskal-Wallis test with Benjamini-Hochberg correction for multiple comparisons. Dunn’s post-hoc test, *p<0.05, **p<0.001, ***p<0.0001

Article Snippet: RNA was extracted from 200 μL of the specimen using Quick RNA Viral kit (Zymo Research) following the manufacturer’s protocol. qPCR for RSV detection and subgrouping was performed using 6.25 μL RNA with Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) and previously published RSV-specific primers and probes .

Techniques: Control, Labeling, Quantitative Proteomics, Activity Assay, Derivative Assay, Gene Expression, Marker

Journal: bioRxiv

Article Title: Unique nasal cell states induced by common pediatric respiratory viruses

doi: 10.64898/2026.04.20.719671

Figure Lengend Snippet: A. Violin plot of normalized single-cell Disease Relevance Scores (scDRS) for childhood onset asthma, adult onset asthma, and height genome-wide association study (GWAS) traits, split by epithelial (top) and immune (bottom) cell type. B. Number of cells with a significant scDRS score at 10% FDR for childhood onset asthma (COA), adult onset asthma (AOA), and height. Cell types are colored as in (A) . C. Cell type-trait association Z-score for childhood onset asthma (COA), adult onset asthma (AOA), and height across epithelial subsets. *FDR adjusted p<0.05. D. Schematic of label transfer approach. Cell type and cell subset annotations were transferred separately from epithelial and immune cells from the USA-Boston cohort to two separate query cohorts. See Methods and Supplementary Table 2 for cohort details. E,G . UMAP of epithelial cells (n=13,514) (E) and immune cells (n=28,342) (G) from all samples in the IND-Kolkata cohort colored by predicted cell type. Cells with prediction score >0.8 are shown. F, H. Spearman correlation of cell subsets from controls ranked by average frequency among epithelial cells (F) and immune cells (H) . X axis, rank in healthy controls in USA-Boston cohort. Y axis, rank in healthy controls in IND-Kolkata cohort. Subsets are colored by cell type of origin as in E, G . I, K . UMAP of epithelial cells (n=12,715) (I) and immune cells (n=5,266) (K) from all samples in the BD-rural cohort colored by predicted cell type. Cells with prediction score >0.8 are shown. J,L. Spearman correlation of cell subsets from RSV cases ranked by average frequency among epithelial cells (J) and immune cells (L) . X axis, rank in RSV cases in USA-Boston cohort. Y axis, rank in RSV cases in BD-rural cohort. Subsets are colored by cell type of origin as in I,K. M . Frequency of select predicted cell types out of all cells per sample in BD-rural cohort, divided by disease group. Participants with <100 total cells were excluded. N. Frequency of select predicted epithelial cell subsets out of all epithelial cells per sample in BD-rural cohort, divided by disease group. Participants with <100 epithelial cells were excluded. O. Dotplot of RSV RNA+ cells in each epithelial subset in all RSV participants in BD-rural cohort. Dot size represents the percentage of cells in the subset that are viral RNA+. Color represents the average viral transcripts (UMI) across all cells in the subset. M,N: Boxplots represent the median (center line), interquartile range (box), and 1.5x the interquartile range (whiskers). Statistical test is Kruskal-Wallis test with Benjamini-Hochberg correction for multiple comparisons. Dunn’s post-hoc test, *p<0.05, **p<0.001, ***p<0.0001.

Article Snippet: RNA was extracted from 200 μL of the specimen using Quick RNA Viral kit (Zymo Research) following the manufacturer’s protocol. qPCR for RSV detection and subgrouping was performed using 6.25 μL RNA with Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) and previously published RSV-specific primers and probes .

Techniques: Single Cell, GWAS

Journal: bioRxiv

Article Title: Unique nasal cell states induced by common pediatric respiratory viruses

doi: 10.64898/2026.04.20.719671

Figure Lengend Snippet: Related to A-H. Quality-control (QC) and prediction metrics for samples in IND-Kolkata cohort. A. Number of cells per sample after selecting high-quality cells (defined as >150 unique molecular identifiers (UMI), >100 unique genes, and <35% mitochondrial reads). Metrics in B-D are restricted to high-quality cells. B. Average number of UMI per cell per sample. C. Average number of unique genes per cell per sample. D. Average percent of mitochondrial reads per cell per sample. E. Distribution of maximum cell type prediction scores within all epithelial cells (left) and immune cells (right). F. Distribution of maximum cell type prediction scores divided by cell type. Cell types are colored as in (G) . G. Distribution of predicted cell types as a percentage of all cells. Each column represents one sample. H-O. Quality-control and prediction metrics for samples in BD-rural cohort. H. Number of cells per sample after selecting high-quality cells (defined as >150 unique molecular identifiers (UMI), >100 unique genes, and <35% mitochondrial reads), divided by disease group. Metrics in I-K are restricted to high-quality cells. I. Average number of UMI per cell per sample by disease group. J. Average number of genes per cell per sample by disease group. K. Average percent of mitochondrial reads per cell per sample by disease group. L. Total number of RSV transcripts (UMI) per sample by disease group. M. Distribution of maximum cell type prediction scores within all epithelial cells (top) and immune cells (bottom). N. Distribution of maximum cell type prediction scores divided by cell type. Cell types are colored as in (G) . O. Dotplot of RSV RNA+ cells in each epithelial subset, divided by sample. Samples with nonzero RSV RNA counts are shown. Dot size represents the number of cells that are viral RNA+. Color represents the total viral transcripts (UMI). H-L: Boxplots represent the median (center line), interquartile range (box), and 1.5x the interquartile range (whiskers). Statistical test is Kruskal-Wallis test with Benjamini-Hochberg correction for multiple comparisons. Dunn’s post-hoc test, *p<0.05, **p<0.001, ***p<0.0001.

Article Snippet: RNA was extracted from 200 μL of the specimen using Quick RNA Viral kit (Zymo Research) following the manufacturer’s protocol. qPCR for RSV detection and subgrouping was performed using 6.25 μL RNA with Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) and previously published RSV-specific primers and probes .

Techniques: Control

Journal: bioRxiv

Article Title: Systematic evaluation of 24 extraction and library preparation combinations for metagenomic sequencing of SARS-CoV-2 in saliva

doi: 10.64898/2026.04.16.719115

Figure Lengend Snippet: RNA from eight nucleic acid extraction kits were assessed based on A) concentration (ng/μL), B) RINe score, and C) representative tapestation RNA gel electrophoresis. (Z1 - Zymo Quick-RNA Magbead, Z2 - Zymo Quick-DNA/RNA Viral Magbead, M1 - MagMAX mirVana Total RNA, M2 - MagMAX Viral/Pathogen, M3 - MagMAX Microbiome Ultra, M4 - MagMAX Viral RNA Isolation, Q1 - QIAamp Viral RNA, P1 - PureLink Viral RNA/DNA)

Article Snippet: The Zymo Quick-DNA/RNA Viral Magbead kit was the only extraction kit that failed to produce detectable RNA via Qubit RNA High Sensitivity Assay Kit in all three positive and negative extracted nucleic acid replicates.

Techniques: Extraction, Concentration Assay, Nucleic Acid Electrophoresis, Isolation

Journal: bioRxiv

Article Title: Systematic evaluation of 24 extraction and library preparation combinations for metagenomic sequencing of SARS-CoV-2 in saliva

doi: 10.64898/2026.04.16.719115

Figure Lengend Snippet: RNA-Seq libraries were created from the eight different nucleic acid extraction methods using three different RNA-Seq library preparation methods: NEBNext Single Cell/Low Input RNA library preparation kit (NEB), Revelo RNA-Seq High Sensitivity library preparation kit (Revelo), and High Sensitivity BRB-Seq RNA library preparation kit (BRB-Seq). Reads were mapped to viral, bacterial, and eukaryotic genomes present in the RefSeq database. The proportion of total reads mapping to SARS-CoV-2 is shown either grouped by A) nucleic extraction method or B) RNA-Seq library preparation method. As a measure of extraction quality, the proportion of total reads mapping to MS2 is shown either grouped by C) nucleic extraction method or D) by RNA-Seq library preparation method. The proportion of E) SARS-CoV-2 reads or F) MS2 reads to total reads is shown with every combination of nucleic extraction methods and RNA-Seq library preparation kits with a darker shade indicating a higher proportion of SARS-CoV-2 or MS2 reads. (Z1 - Zymo Quick-RNA Magbead, Z2 - Zymo Quick-DNA/RNA Viral Magbead, M1 - MagMAX mirVana Total RNA, M2 - MagMAX Viral/Pathogen, M3 - MagMAX Microbiome Ultra, M4 - MagMAX Viral RNA Isolation, Q1 - QIAamp Viral RNA, P1 - PureLink Viral RNA/DNA). Statistics were calculated by a One-Way ANOVA, followed by Tukey’s HSD post-hoc test. Asterisks above connecting brackets indicate significant differences between those two specific groups. * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001.

Article Snippet: The Zymo Quick-DNA/RNA Viral Magbead kit was the only extraction kit that failed to produce detectable RNA via Qubit RNA High Sensitivity Assay Kit in all three positive and negative extracted nucleic acid replicates.

Techniques: RNA Sequencing, Extraction, Single Cell, RNA Library Preparation, Isolation