Cell free / circulating tumor DNA Analyses

Cf / Ct DNA Analyses

About

At MOMA we have a long track record of reliable identifying and quantifying circulating tumor DNA (ctDNA) by either NGS or droplet digital PCR (ddPCR).
All our services are offered as non-profit for research groups and clinical departments at universities or hospitals in Denmark to help facilitate cfDNA and ctDNA analyses.
The cfDNA Analyses team is part of the MOMA NGS Core Center.
Our team of highly trained personnel provides guidance and advice for sequencing and ddPCR projects.

Equipment

Our fully equipped lab, including automation using liquid handling robots, support all common sequencing applications as well as the development of novel methodologies and protocols. The ddPCR equipment consists of the QX200 Droplet Digital PCR (ddPCR) system which provides absolute quantification of target DNA molecules for probe-based digital PCR applications.

Data analysis

For both NGS and ddPCR cfDNA/ctDNA analyses MOMA will perform the bioinformatics data analysis resulting in in a sample being called either positive or negative.
The number of ctDNA molecules per sample and variant allele frequency will be provided.

Publication Policy

The use of the services provided by the NGS Core Center in publications requires acknowledgement of the center.
A suggested acknowledgement is: Library preparation / Next generation sequencing/ ddPCR cfDNA/ctDNA analyses was performed by the cfDNA Analyses team, NGS Core Center, Department of Molecular Medicine, Aarhus University Hospital, Denmark.

cfDNA Analyses

Targeted sequencing

  • TruSight Oncology 500 ctDNA [1]
  • Sporadic  colorectal cancer (APC, BRAF, FAM123B, FBXW7, KRAS, NRAS, PIK3CA, SMAD2, SMAD4, SOX9, and TP53)

Multi-assay detection

  • Trimeth (Validated methylation marker assays for colorectal cancer patients) [2]

Tumor informed single target detection

cfDNA related services

Targeted sequencing

  • Whole exome Sequencing (cancer and buffy coat DNA) [3].
  • TruSight Oncology 500 High-Throughput (TSO500 HT) [4]  (cancer DNA)
  • Sporatic colorectal cancer (targeted sequencing of colorectal cancer and buffy coat DNA) (APC, BRAF, FAM123B, FBXW7, KRAS, NRAS, PIK3CA, SMAD2, SMAD4, SOX9, and TP53)

Single target screening/detection

DNA extractions

  • cfDNA extraction from 2-8 mL plasma
  • DNA extraction from fresh frozen, FFPE cancer tissue
  • DNA extraction from buffy coat

Instrumentation 

Illumina Sequencing 
NovaSeq 6000
NextSeq 500
MiSeq

Please see www.illumina.com for additional information on performance and specifications.

Quality Control
2 x Agilent Tapestation 4200
2 x Qubit 3.0
2 x BioTek Synergy LX Platereaders
1 x Lunatic Unchained Labs Dropsense

Library preparation
2 x PerkinElmer Sciclone G3 NGS – automated liquid handling system
1 x Covaris E220 Evolution
1 x 10x Genetics Chromium Controller

Biorad ddPCR system [5]
1 x QX200 AutoDG Droplet Digital PCR System
1 x PX1 PCR Plate Sealer
1 x PX1 PCR Plate Sealer
2 x QX200 droplet reader


 

ddPCR reference assays and quality control
cfDNA quantification assays
cfDNA spike in quantification assays
High molecular weight DNA marker
Positive controls
Negative controls
2 x Agilent Tapestation 4200

Please see bio-rad.com for additional information on performance and specifications [5].
 

Other
2 x Applied Biosystems 3500 XL Series Genetic Analyzers


Typical Workflow

Please, see the document Sample Requirements and Data Analysis for further details. 
Prior to shipping samples please see the Shipping information and complete the Sample sheet template (Excel format).

Procedure

Please contact us for more information about our services and prices.

We will arrange a short meeting to discuss your project in detail. This will cover the approach, targeting, library preparation and data handling. Our scientists will assist you in identifying the design that fits your project best.
A customer contract including a time schedule is required before your samples can be processed.
After completion of the deep sequencing analyses, we will perform the initial data processing. The data will be handed over to you personally as agreed upon.

References

1. https://emea.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500-ctdna.html
2. Sarah Ø.J. et. Al; Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study. Clin Epigenetics. Published 2019 Nov 14. doi:10.1186/s13148-019-0757-3)
3. https://www.twistbioscience.com/products/ngs
4. https://emea.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500-ht.html
5. https://www.bio-rad.com/en-dk/product/qx200-droplet-digital-pcr-system?ID=MPOQQE4VY