simplSEQ, Inc., a spinout of Murietta Genomics focused on technology for isolating and analyzing circulating nucleic acid to better understand health and disease, presented a poster, “A Novel cfDNA Isolation Procedure: An Enzymatic Modification Method Results in Higher Yields, Simplied Procedure, and Minimized Size Bias,”at the 2021 Association for Molecular Pathology (AMP) annual meeting showing the value of the simplSEQ technology in isolating DNA and RNA from whole blood. The data presented showed that the patented simplSEQ method was able to isolate more DNA, eliminate size biases, and isolate both DNA and RNA from a human whole blood sample.

Cell-free DNA (cfDNA) is becoming an increasingly common input in liquid biopsy-based molecular diagnostics for wide-ranging applications including cancer diagnosis, cancer recurrence and treatment monitoring, transplant monitoring, and non-invasive prenatal screening, among others. 

Despite the growing prevalence of these applications, clinical laboratories are often challenged to mitigate effects of hemolysis in order to maximize the opportunity to find cfDNA signal amid other DNA within a sample and are typically unable to conserve sample in order to perform other assays, such as RNA sequencing. Despite advances in preanalytical technology, there has been limited innovation in nucleic acid isolation, and failure to isolate enough DNA from which to prepare a sequencing library remains a challenge across liquid biopsy applications.

The simplSEQ novel extraction protocol adds modified nucleotides to the 3’ ends of the nucleic acids which are bound to a solid matrix. It then uses template independent polymerase Terminal Transferase to extend the 3’ ends of DNA and RNA and magnetic beads enable attachment of the affinity labeled cfDNA to the matrix. When the beads are washed and magnetically separated, the isolated, bead-attached cfDNA can be used either directly in PCR based assays, or cDNA (complimentary DNA) copies can be created for downstream whole-genome or targeted next-generation sequencing assays. 

“Next-generation sequencing assays performed using the simplSEQ protocol with DNA attached to beads produced better quality data with an improved percentage of aligned reads, on-target reads, and coverage uniformity,” says Brandon Young, cofounder and Chief Scientific Officer at simplSEQ.  “Since these quality metrics correlate to enhancing sequencing depth, laboratories following this protocol can expect to reduce overall sequencing costs, and improve their rates of successfully completing diagnostic tests.”

This method also enables improvements to turnaround time resulting from beginning the sample extraction process while the sample is still in transit, reducing labor and consumables costs due to a simpler and more reproducible workflow, improving data quality and nucleic acid yield, and enabling laboratories to perform multiple DNA and RNA extractions from a single sample.

“Clinical laboratories need solid protocols that are easy to teach and offer consistency across all laboratory operators,” said John Spinosa, M.D., Chief Medical Officer at simplSEQ and formerly a practicing pathologist. “The simplSEQ workflow offers many workflow advantages over other methods of cell-free DNA extraction especially speed, simplicity, reproducibility, which are critical as liquid biopsy becomes more common.”