Library-free, targeted sequencing of native gDNA from FFPE samples using Hyb & SeqTM technology – the hybridization based single molecule sequencing system

DNA sequencing is a promising tool for personalized medicine, but widespread implementation is hindered by complexities in sample preparation and sequence analysis. Hyb & Seq technology is a library-free, amplification-free... [ view full abstract ]

DNA sequencing is a promising tool for personalized medicine, but widespread implementation is hindered by complexities in sample preparation and sequence analysis. Hyb & Seq technology is a library-free, amplification-free single molecule sequencing technique that uses cyclic nucleic acid hybridization of fluorescent molecular barcodes onto native targets.  Hybridization-based sequencing enables the simplest sample to answer workflow, with almost no manipulation of the input material. 

Here, we provide an end-to-end Hyb & Seq demonstration that consist of i) sample preparation including simple, rapid gene capture from formalin-fixed paraffin embedded (FFPE) tissue, ii) library-free targeted sequencing of actionable oncogenic mutations, and iii) robust primary data analysis using the Hyb & Seq assembly algorithm (ShortStack^TM) leading to accurate variant calling.  Hyb & Seq targeted sequencing of oncogenes was performed on NanoString’s prototype Hyb & Seq system. This work represents the continued, substantive progress of Hyb & Seq chemistry, first introduced at 2016 AGBT.

Highlights of Key Results:

• Total processing time from FFPE curls to start of sequencing was under 60 minutes, with total hands-on time of less than 15 minutes
• Current un-optimized target capture of one to three FFPE curls (10 microns thickness) was sufficient for sequencing with no PCR amplification
• Four-hundred cycle experimental data presented

Hyb & Seq’s simplicity, flexibility, and accuracy offers an ideal sample-to-answer solution for the sequencing lab.  Hyb & Seq technology is currently for research use only and is not for use in diagnostic procedures.