ASHG 2025 — Boston, MA
Multi-Site Benchmarking Studies For PRECYSE , A Novel DNA/RNA Single Molecule Resolution Imaging And Analysis QC Platform For Long And Short Read NGS Libraries
Here we present data from a comprehensive benchmarking study for a novel benchtop biomolecule imaging and analysis platformc PRECYSE, from Evizia, that enables rapid visualization and analysis of DNA from 100 bp to >250,000 bp. Our study was conducted at six leading genomics core laboratories in the US and tested the performance in analyzing over 200 DNA and RNA samples of various types and qualities during library preparations for Next Generation Sequencing (NGS). Samples tested included long and short read DNA/RNA libraries, human gDNA from blood, saliva and FFPE, ultra-high molecular weight DNA for ONT, cfDNA size selections, polymerase and helicase bound libraries, and sheared gDNA.
Quality control (QC) in complex NGS workflows is a critical, established and clinically required step in the preparation of nucleic acid samples for analysis. However, existing QC methodologies such as gel electrophoresis have failed to keep pace with rapid advancements in sequencing technologies, impeding broader clinical adoption. Evizia aims to consolidate the complex instrument and kit offerings utilized today for NGS library QC with a single desktop instrument and consumable type, at lower cost, with no cold chain components, simplified workflow, and superior single molecule direct visualization and analysis.
Jason Reed1, Katia Sol-Church2, Kelly Cribari3, ChewYee Ngan4, Yongjun Huang4, Sophie Low5, Michelle Cipicchio5, Evelyn Ng6, Jack Wheeler6, Guilin Wang6, Karolina Aberg7, and Brian Patterson1
1Evizia, Inc., Richmond, VA, 2University of Virginia School of Medicine, Charlottesville, VA, 3Harvard University, Cambridge, MA, 4The Jackson Laboratory, Farmington, CT, 5The Broad Institute of Harvard and MIT, Cambridge, MA, 6Yale University School of Medicine KMSR, New Haven, CT, 7Virginia Commonwealth University, Richmond, VA
Figure 1. PRECYSE Workflow Overview.
A. Samples are diluted in water to 10 – 1600 pg/uL (range depends on approximate sizing required) and combined 1:1 with EZbuffer. 2 µL is added to each ‘spot’ of an EZchip followed by a short room temperature incubation. Washing, drying and heating is automated before loading the chip for scanning. Different sample types can be run togetether on a single multi-plexed EZchip, no ladders or standards required. B. PRECYSE invokes proprietary scanning based on HSAFM (High Speed Atomic Force Microscropy) technology for direct imaging from 100 bp to 250+ kbp with single molecule resolution. Raw image ‘frames’ are viewed in real time before onboard machine learning analysis software traces DNA molecules for exact molecule sizing and histogram and/or scatter plot generation.
Figure 2. gDNA Analysis and Comparison to Gel Electrophoresis
A. Stock female Promega gDNA scanned by PRECYSE at VCU. One example frame shown. Blue outlines are the PRECYSE software tracing and sizing analysis. Scatter plot of sizing for 227 molecules shows a long distribution of larger molecules out to ~200 kb, indicative of intact high quality gDNA. B. gDNA extracted from albatross provided by Harvard University. PRECYSE imaging shows ssDNA and small particulates in the sample. Sizing analysis of 154 molecules indicates good quality gDNA extending out to ~111 kb. Corresponding Agilent Femto analysis shows a similar size distribution but severely underrepresents the percentage of smaller molecules due to mass-based fluorescence. The non-linear separation and data display of gel electrophoresis makes sizing of larger DNA difficult. C. Human gDNA extracted from FFPE and analyzed by PRECYSE at The Jackson Laboratory. PRECYSE sizing analysis of 849 molecules generated a median size of 1.7 kb with sizes ranging from 212 to 19,784 bp, indicative of degraded/sheared gDNA. Extremely small DNA or particulates, and cross-linked DNA (intense thicker white molecules), is shown. The Agilent TapeStation electropherogram of same FFPE gDNA sample produced sizing from 250 to >48,500 bp (max instrument resolution). This larger incorrect sizing is presumably due to the highly crosslinked nature of FFPE gDNA.
Table 1. PRECYSE Comparison to Automated Gel Electrophoresis Systems
Figure 3. Long Read NGS Library QC
PacBio library provided by The Jackson Laboratory. A. Example raw PRECYSE image of several molecules within one frame. B. PRECYSE sizing analysis of PacBio Library and 551 molecules spanning multiple image frames. Median size of 15.1 kb and mode at 14.7 kb measured. C. HiFi read length distribution from the PacBio Revio showed a median of 13.3 kb and mode of 11 kb. Femto analysis (data not shown) produced a median size of 17.4 kb and Mode of 15.9 kb. PRECYSE analysis more closely aligns with the HiFi read distribution. Note that the non-HiFi reads are not shown.
Figure 4. Short Read NGS Library QC
A. Example partial frame image from PRECYSE of a short read Illumina library. B. PCR-free mouse Illumina library provided by The Jackson Laboratory. Sizing analysis of 1209 molecules generated a median of 533 bp and mode at 493 bp. C. Agilent TapeStation electropherogram of same PCR Free Illumina library showed a mode of 1200 bp. Slow migration of molecules on the Tapestaion due to Y-adaptors overestimates true sizing. PRECYSE sizing is not impacted by adaptors and secondary structure, and accurately sizes the PCR Free Illumina library. D. Human cfDNA sample analyzed at Yale University. Sample is post Pippin Prep size selected targeting 250-450 bp (Sage Science). E. Corresponding TapeStation electropherogram of same cfDNA sample produced a median of 362 bp. TapeStation and PRECYSE primary sizing modes align well. PRECYSE was able to show improved sizing granularity with a second mode at ~620 bp, and larger molecules extending out to >1400 bp. Sizing of small DNA < 2 kb with PRECYSE requires < 50 pg of material. Sub pg analysis is possible with extended scanning times.