APPLICATIONS
PRECYSE is a general-purpose molecular biology instrument for all labs, replacing applications currently performed by electrophoresis. It offers superior speed, accuracy, precision, lower input requirements, and direct molecular analysis without the need for cold-chain dyes or comparative ladders.
Broad range of applications
PRECYSE currently supports DNA sizing with a vastly improved solution for NGS and Synthetic Biology QC versus electrophoresis.
- High Speed Atomic Force Microscopy (HSAFM)
- Direct physical imaging and software analysis
- NGS QC, repeat expansion, protein-DNA binding, and more
- 100bp to 500kbp+ DNA sizing
- Single molecule 2D/3D visualization and resolution
- Single consumable EZchip for all applications
- Simple sample prep with no cold chain consumables
- Fast analysis time from 1-6 min per sample
Short Read NGS QC
Illumina DNA prep (Nextera DNA Flex) library
Figure. Zoom-in of PRECYSE raw image of sample and DNA library molecules (2 uL at ~1 pg/uL). Total sample scan and analysis time of ~ 1 min.
Figure. PRECYSE image analysis and sizing histogram of 835 molecules generated a mean size of 454 bp with additional resolution of molecules >1000 bp.
Figure. Competitor instrument TS electropherogram utilizing a D5000 HS kit produced a mean of 406 bp with RFU peak at 300bp.
PRECYSE histograms are based on molecule absolute sizing counts instead of electrophoresis mass-based fluorescence intensity. Small and large molecules are counted equivalently, providing a direct comparison to sequencing read length output distributions.
Additional advantages of PRECYSE include speed, no cold chain consumables, direct imaging without sizing ladders or fluorescence dyes, no upper limit to DNA sizing, observation of secondary structure and contaminants, and drastically improved precision and accuracy.
Long Read NGS QC
PacBio Revio human DNA library
Figure. Example zoom-in image from PRECYSE showing DNA molecules and sizing in kbp.
Figure. Competitor Femto instrument analysis produced RFU peak of 15.9 kbp and mean of 17.4 kbp.
Figure. PRECYSE analysis of 162 molecules generated a mean of 15 kbp.
Figure. PacBio Revio results of sequencing produced a mean insert size of 14.0 kbp.
Summary: Better sizing correlation between PRECYSE and PacBio Revio read length output was observed. Electrophoresis of larger DNA consistently overestimates the true sizing of DNA because the technology relies on indirect calculations based on mass- based fluorescence (output is highly biased toward larger molecules). PRECYSE counts small and large molecules equivalently, providing a direct comparison to sequencing read length output distributions.
Oxford Nanopore (ONT) human DNA library
Figure. Example molecules from PRECYSE scan with analyzed sizes (kbp).
Figure. Competitor Femto analysis produced RFU mean of 43.7 kbp.
Figure. PRECYSE analysis of 41 molecules generated a mean of 12.2 kbp.
Figure. ONT sequencing read results produced a N50 of 14.6 kbp.
Summary: Significantly better sizing correlation between PRECYSE and ONT N50 read length output was observed. Electrophoresis of larger DNA consistently overestimates the true sizing of DNA because the technology relies on indirect calculations based on mass- based fluorescence (output is highly biased toward larger molecules). The competitor Femto grossly overestimated the true mean library size by nearly 30 kbp. PRECYSE counts small and large molecules equivalently, providing a direct comparison to sequencing read length output distributions.
gDNA NGS QC
Non-sheared human gDNA
Figure. Example zoomed-in image of DNA molecules before software tracing analysis. PRECYSE mean sizing of 9.8 kbp from 139 molecules. Total scan and analysis time of ~6 min.
Figure. Competitor Femto electropherogram produced a mean RFU intensity of 55.5 kbp.
Figure. PRECYSE sizing histogram with a 9.8 kbp mean from 139 molecules analyzed. Total scan and analysis time of ~6 min.
Summary: PRECYSE shows ~36% of the gDNA molecules are 2-3 kbp in size and only ~9% are >20 kbp. Direct size distributions of larger DNA molecules required for long read sequencing library preps is not easily inferred from RFU electropherograms and mean/mode values based on mass consistently overestimate true sizing.
Sheared gDNA NGS QC
Figure. Example zoomed-in image of DNA molecules with software tracing and sizing analysis shown in kbp.
Figure. Competitor Femto electropherogram produced a mean RFU of 18.2 kbp and max RFU of 15.5 kbp.
Figure. PRECYSE histogram analysis of 115 molecules had a mean size of 12.6 kbp. Total image and analysis time ~6 min.
Summary: PRECYSE shows ~38% of the gDNA molecules are 2-6 kbp in size. Femto electropherogram based on RFU does not properly show the distribution of molecules <10 kbp and overestimates true sizing.
NGS QC Specifications and Comparison
DNA Mapping
Figure. Long and short-range sequence mapping with customizable Cas9 nano-labels
Identifying sequences with CAS9
Figure. DNA map and corresponding PRECYSE image of Cas9-labeled BRCA1 amplicon. Red tick marks represent locations of the Alu repeat-targeted sgRNA-binding sites.
Figure. Measured vs expected position of the Cas9 labels from BRCA1, TERT-HER2 and ladder constructs indicating accurate linear sizing out to >200kbp. Mikheykin et. al., Nature Communications, 2017.
Figure. Repeat expansion can be measured by PRECYSE.
Repeat expansion disease (FMR1)
Figure. FMR1 (fragile X syndrome) repeats were measured utilizing Cas9 excision and gel based size selection (Sage Science) before labeling and imaging with PRECYSE.
Protein Binding
Figure. Enzyme binding to DNA can be observed with PRECYSE. Shown here are 2 NGS library preps ready for sequencing.
Figure. Oxford Nanopore Helicase protein is shown bound to a large DNA molecule. The green color represents a significant height difference due to the large enzyme structure vs the lighter blue DNA molecule.
Figure. PacBio polymerases are shown in 3D represented by the higher red structures. 2 of the 3 PacBio polymerases observed are bound to the DNA.
Contaminants and Secondary Structure
Figure. Example gDNA samples imaged by PRECYSE.
Figure. Insect gDNA sample provided by a collaborator contained a high concentration of dsDNA molecules with ssDNA tails, secondary structure that cannot be observed by electrophoresis.
Figure. 3D zoomed-in rendering of insect gDNA 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.