High-throughput Genomic Analysis of Urine Samples Using Pixelated-Ultrasoundto Improve Urine Biopsy Workflow
Martha Evans-Holm1, Nicolai Lumbres1, Rwik Sen2, Patricia Montilla-Perez2, Marc A. Paradise2, Peter Lentz1
1
Convergent Genomics, South San Francisco, CA, 94080, USA 2Active Motif, Inc., Carlsbad, CA 92008, USA

Poster # 5328

Schematic of Workflow for Urine Genomic
DNA Analysis Using PIXUL® Sonicator

Abstract
The challenge addressed here is an improvement to sample preparation of liquid biopsy fromcancer patients for sequencing. We present the advantages of a novel pixelated ultrasoundtechnology which significantly improves sample-preparation in a very fast, economical, andhigh-throughput manner with very consistent output between samples.
The technology works consistently for both low and high cell numbers which is a critical factor
for heterogeneous samples from patients with no cross-contamination. In conclusion, wepresent and improvement in the workflow of non-invasive liquid biopsy using urine samples.
This improvement will greatly enable unique fingerprinting of an individual’s cancer or cancer
risk across large cohorts, with the potential for integration in automation workflows.

High-throughput
sample processing

Treatment of sample
for preservation*

Results: Superior Consistency in Output using PIXUL® sonicator

Next-Generation
Sequencing

Bioinformatics
Cloud Computing
Machine Learning Algorithms

Shipment of sample
to facility
Clinical Reporting
Clinical Management of Disease​

Storage* of samples

Therapeutic Development*US Patents
US-20220213558-A1
US-11421217-B1
US-20220389405-A1
US-20230040907-A1

PIXUL® SonicatiorCells

Collection ofurine samples

FFPE
Plant Samples

VirusTissues

Genomic DNA

Chromatin
Microbial Cells

Enabling Epigenetics ResearchEquipment

Average Size (base pairs)

PIXUL 30 min
PIXUL 48 min
Non-PIXUL

248
224
168, 491 (2 peaks)

Figure 3. Comparison between PIXUL® and non-PIXUL sonicator

Advantages⊲ Obtain ideal fragment sizes, consistently across high-throughput samples in multiple batches forlibrary preparation⊲ Target enrichment by flexible programming⊲ Consistent, fast, economical, low input requirement, user-friendlySamples processed with PIXUL® consistently exhibited more thorough fragmentation, with a single majorpeak, and lesser variation whereas the samples processed by the non-PIXUL alternative showed multiple
major peaks with broader variation. Additionally, PIXUL® produced a narrower overall range of DNA
fragment sizes than the non-PIXUL alternative.

Figure 1. Schematic for processing high-throughput samples derived from urine and integration of PIXULin the protocol leading to overall improvement of workflow, and hence clinical management of disease.
Proprietary steps of sample treatment in the protocol are covered by US patents as indicated.

Materials and Methods​
Sample collection and processing: Healthy human donor urine sample collection is a manual process
where the donor collects their sample in a classic urine collection cup. It is then transferred to Vacutainer
tubes. Sample processing is centered around a genomic DNA extraction method. The urine samples
undergo extraction and nucleic acids are stored at 4°C or at -20°C for downstream analysis.
PIXUL Sonication: PIXUL® Multi-Sample Sonicator (Active Motif Catalog No. 53130) uses a transducerlens assembly capable of focusing ultrasound, without auditory hazard. Individual samples are pipetted
into specific wells of commercially available 96-well U-bottom polypropylene microplates (Corning CatalogNo. 3799) which is then sealed and loaded into PIXUL® for processing. A high-power amplifier drives thetransducer array, while an in-built Peltier cooling system is used to reduce heating of the samples. The
built-in computer controls the ultrasound pulse parameters (number of cycles, treatment configurations andprocessing time). The device is capable of processing 96 samples at one time. It also allows the user toassign different sonication settings in each column (total 12) of a 96-well plate using the touch-screen userinterface. The following recommended default settings were used:

Workflows
Genomics
Metabolomics

Proteomics
Chemistry

Transcriptomics

Pharmaceutical

Epigenetics

Mesoscale Discovery⊲ ChIP-Seq

PIXUL®’s consistency through fragmentation across a 96-well plate. Instruments often exhibit variability inperformance across a plate due to engineering limitations, such as edge-effect, making this an essentialconsideration. Once again, PIXUL® performed exceedingly well in this regard.

Acknowledgments and References⊲ Cytokine Profiling⊲ Hi-C

1. Crocetto F., et al. 2022 Liquid biopsy in bladder cancer: State of the art and future perspectives
Critical Reviews in Oncology/Hematology 170:103577⊲ DNA &RNA Methylation

We compared the PIXUL sonicator with an alternative non-PIXUL high-throughput sonicator, samples
were loaded into the manufacturer’s proprietary consumable labware and processed. The manufacturer’s
recommend protocol was followed.

Introduction
The most common malignancy of the urinary tract is bladder cancer, ranking as the 9th mostcommon cancer worldwide and 2nd most common cause of death due to genitourinary
tumors in the United States [1]. Considering the statistics, recurrence and costs associatedwith treatment bladder cancer and other urological cancers, it is important to develop
efficient methods to screen patients for cancer risk assessment and prevention. Several
factors contribute to risk factors including age, ethnicity, gender, genetics and environmental
carcinogenic exposure. A crucial step for the success of NGS-based experiments is the
preparation of good quality libraries which will undergo sequencing and subsequent analysis.
Generating a good quality library is a major bottleneck because DNA fragments in a goodquality library consistently need to belong to a specific size range which is difficult to obtainusing traditional library preparation methods. Further, reproducibility, consistency, and rigor
across high-throughput samples are required for screening and risk assessments to prevent
cancers. Overall, it is very challenging to consistently fragment DNA to a specific size range withgood reproducibility across high-throughput samples in a fast and economical manner requiring
low input material, all at the same time. Hence, we present pixelated ultrasound (PIXUL®)
technology [2, 3] which helps to obtain DNA fragments with sizes that qualify for good libraries
consistently across high-throughput samples, i.e. 96-well plate in a fast and economical manner.

activemotif.com

Figure 4. High Consistency between wells of PIXUL® sonicator

DNA Fragmentation Analysis: Fragment Analyzer from Agilent Technologies and its compatible kit
for DNA fragment analysis were used to test the fragmented DNA samples using the manufacturers
recommended protocol. The included ProSize software was used fordata analysis.

2. Bomsztyk K., et al. 2019 PIXUL-ChIP: integrated high-throughput sample preparation andanalytical platform for epigenetic studies Nucleic Acids Research 47(12):e69
3. Mar D., et al. 2024 A High-Throughput PIXUL-Matrix-Based Toolbox to Profile Frozen andFormalin-Fixed Paraffin-Embedded Tissues Multiomes Laboratory Investigation 104(1):100282
4. PIXUL® is sold under an exclusive license to patents owned by Matchstick Technologies Inc.
and University of Washington, specifically Matula, T.J.; Bomsztyk, K.; Darlington, G.P., Maxwell,A.D.; MacConaghy, B.E., Reed, J. “Ultrasound system for shearing cellular material”. US PatentUS10809166. European Patent EP3169451

Summary
1. The results prove the efficacy of the technology in providing precise DNA fragments from limited
input samples in a fast, consistent, and economical manner on a high-throughput platform.
Figure 2. Left, PIXUL easy set up. Right, donor samples loaded across the 96-well, emphasizing theedges, corners, and center of the plate so that sonication in wells at diverse locations on the plate can becompared to test well to well consistency.

2. PIXUL® provides the flexibility of assigning 12 different sonication programs to the 12 columns of
the 96-well plate.
3. PIXUL® is a novel technology which provides a consistent, high-throughput, fast and economical
method of enabling urinary liquid biopsy.

Learn more at activemotif.com/pixul

Contact:rsen@activemotif.com

tech_service@activemotif.com