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Extracting HMW DNA from saliva for HiFi sequencing applications

Laboratory Genetics and Genomics
  • Primary Categories:
    • Laboratory Genetics
  • Secondary Categories:
    • Laboratory Genetics
Introduction:
Saliva is an attractive DNA source for large-scale genomic and clinical research studies due to its non-invasive collection and ability to be self-administered. However, concerns about DNA quality and yield have limited its use with long-read sequencing, where high-quality, high-molecular-weight (HMW) DNA is key for optimal yield and performance.

As a sample type, saliva is stable at room temperature in DNA Genotek™ Oragene™ devices. DNA from saliva samples is generally ~75−95% human (whereas DNA from buccal samples can be as low as 10% human). The majority of DNA isolated from saliva comes from white blood cells (not from buccal epithelial cells). In this proof-of-concept study, we demonstrate that high quality PacBio® HiFi sequencing results can be obtained from DNA extracted from saliva collected into DNA Genotek Oragene devices and extracted using the Nanobind® PanDNA or CBB kits.

Methods:
Saliva samples were self-collected by 17 individuals using Oragene devices, with six matched blood samples also collected. HMW DNA was extracted from 500 µL of saliva and 200 µ L of blood with the Nanobind kits. Following lysis, the DNA binds to Nanobind disks which shield bound DNA from damage during extraction, resulting in HMW DNA. DNA was then size-selected using the SRE kit and sheared to 15-20 kb with robotic pipette shearing. Automated PacBio library preparation was performed on the fully automated Hamilton NGS STAR system. Samples were prepared using SPRQ™ chemistry kits and sequenced on a single SMRT® Cell on the Revio™ system. HiFi reads were mapped against the human genome reference, with variant calling performed using SMRT® Link analysis pipelines. Unmapped human reads could be used to classify the saliva microbiome.

Results:
With Nanobind, we recovered 1 to 45 µ g of pure HMW DNA from saliva samples collected in Oragene devices. For the 17 samples, we obtained 104 Gb to 135 Gb of HiFi data resulting in 20x to 40x coverage per genome, sufficient for comprehensive WGS variant detection. Saliva and blood paired samples were assessed for concordance in genome coverage quality and variant detection between sample types within each donor. Taxonomic profiling analysis was done on unmapped human reads and bacteria were identified. Over 90% of classified reads are from the five following phylum: Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Fusobacteria.

Conclusion:
This method demonstrates HiFi sequencing performance from saliva samples collected with Oragene devices and extracted using Nanobind kits. It shows that saliva can be a viable alternative to blood for obtaining high-quality long-read sequencing data. The non-invasive nature and ability to mail collection kits enables more efficient, cost-effective recruitment for large studies and facilitates the use of long-read sequencing in precision medicine initiatives and population genomics studies that leverage saliva samples.

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