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The Jackson Labs Long Read Workshop 2024

High Speed Atomic Force Microscopy (HSAFM) for analysis of gDNA and long read sequencing library construction QC

Evizia introduces PRECYSE, a DNA imaging, mapping and analysis instrument, and its application for short and long read NGS sample and library QC. Current market leading instruments are based on electrophoresis, a well-established technology with significant sensitivity, precision and sizing limitations. PRECYSE invokes high speed atomic force microscopy (HSAFM) for direct imaging of DNA from 100 bp to 500+ kb with single molecule resolution. A user replaceable probe on the end of an AFM cantilever mechanically scans a consumable surface containing biomolecules such as DNA measuring forces between the probe and sample to generate a 2D and 3D topographical images down to nanometer resolution.

Andrey Mikheikin[1], Kelly Cribari[4] and Jason Reed [1,2,3]

[1] Evizia, Inc., Richmond VA, [2] Physics Department, Virginia Commonwealth University, Richmond, VA, and [3] Massey Comprehensive Cancer Center, Richmond, VA, and [4] Bauer Sequencing Core, Harvard University, Cambridge MA

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DNA molecule sizing precision illustration
Figure 1.  PRECYSE is a desktop instrument based on HSAFM. Biomolecules such as DNA and RNA are deposited and dried with heat onto an atomically flat surface (proprietary disposable ‘chip’ from Evizia) and visualized via contact mode scanning of an AFM probe attached to a cantilever. Up to 96x 2-3 uLof <1 pg/uL samples can be scanned per chip at 1-6 min per sample (depending on the size of the molecules).
PacBio SMRTbell Library QC Illustration
Figure 2.  PacBio SMRTbell Library QC of Human DNA sample provided by The Jackson Laboratory post SRE and Blue Pippin cleanups (A) Example of several molecules imaged by PRECYSE (B) Femto Pulse analysis produced a mean library size of 17.4kb. (C) PRECYSE analysis of 162 molecules generated a mean of 15.0kb. (D) PacBiosequencing generated HiFi length of 14.0kb from 3.5M reads and 48.7 Gb yield. Mean sizing from PRECYSE correlated better to PacBio mean insert size.
gDNA sizing illustration
Figure 3.  Analysis of Red Milkweed Beetle gDNA provided by Harvard and sizing comparison to the Agilent Femto Pulse. (A) Zoomed-out example image. (B) Zoomed-in example scans of 9-20 kb gDNA molecules in sample. (C) Femto pulse analysis showing mode (max RFU) of 12 kb. (D) PRECYSE analysis of 257 molecules showing a high concentration of 1.5kb fragments along with a distribution of larger gDNA >30kb not visible by the femto trace. Femto and PRECYSE mean sizes were similar at 13.1 and 13.0 kb respectively..
[1] Technical information sourced from vendor materials available through public sources [2] US list price [3] Per sample with max throughput, US list price [4] Based on 1x 100 bp DNA molecule [5] Best possible from all available kit specifications published
gDNA contaminants and secondary structure Illustration
Figure 4.  Example gDNA samples imaged by PRECYSE. (A) 3D zoomed-in rendering of an unknown insect gDNA sample provided by Harvard that showed a large amount of unknown non-DNA contaminant easily viewed as taller objects bound to the surface compared to DNA molecules. This contaminant was not visible by electrophoresis. (B) Another unknown insect gDNA sample provided by Harvard that contained a high concentration of dsDNA molecules with ssDNA tails, secondary structure that could only be visualized by PRECYSE.
Table 1: Comparison of important NGS QC usability specifications of PRECYSE vs market leading automated electrophoresis platforms. Best performance highlighted in green.
Conclusion: PRECYSE is a first of it’s kind direct imaging desktop instrument and consumable solution designed specifically for DNA imaging and analysis. For NGS QC it addresses limitations of electrophoresis through vastly improved sensitivity, accuracy, and precision, via direct single molecule analysis with a simple workflow that eliminates cold chain reagent usage. For gDNA and PacBio libraries we demonstrate better sizing detail and accuracy with no upper limit, visibility to potential contaminants, and secondary structures. Through better NGS QC PRECYSE will save time, money and improve long read sequencing results.