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: Positive and negative contrived saliva samples were created to test eight nucleic acid extraction methods for mNGS RNA testing. A) SARS-CoV-2 negative saliva samples were collected and pooled from 96 individuals. MS2 phage was added to the pool at a final concentration of 12,500 copies/μL. The pooled saliva and MS2 phage samples were split into positive and negative samples. Heat-inactivated SARS-CoV-2 was added to the positive sample with a final concentration of 5,000 copies/μL. B) Saliva samples were then extracted using one of eight different extraction kits, and RNA-Seq libraries were generated with one of three library preparation kits. The extracted nucleic acid was assessed for quality and quantity using TapeStation and Qubit. RNA-Seq libraries were then sequenced, and the SARS-CoV-2 and MS2 reads were quantified.

Article Snippet: We generated contrived positive saliva samples by pooling 96 SARS-CoV-2 PCR-negative saliva samples from healthy individuals, adding MS2 phage (Varizymes, 6000L) with a final concentration of 12,500 copies/μL, and adding Heat-inactivated SARS-CoV-2 (ATCC, VR-1986HK) with a final concentration of 5,000 copies/μL.

Techniques: Extraction, Concentration Assay, RNA Sequencing, Generated

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: We generated contrived positive saliva samples by pooling 96 SARS-CoV-2 PCR-negative saliva samples from healthy individuals, adding MS2 phage (Varizymes, 6000L) with a final concentration of 12,500 copies/μL, and adding Heat-inactivated SARS-CoV-2 (ATCC, VR-1986HK) with a final concentration of 5,000 copies/μL.

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