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NGS Masters: Whole-Genome Sequencing of cfDNA and ctDNA
Enhance CNV detection with Collibri PS DNA library prep kits
Whole-genome sequencing is suitable for the study of disease-specific genomic abnormalities from cfDNA. The Invitrogen Collibri PS DNA Library Prep Kit for Illumina Systems enables consistent and reproducible cfDNA library preparation from human plasma samples.
Optimized protocol captures short cfDNA fragments
Circulating cell-free DNA (cfDNA) is becoming an increasingly important research sample to investigate circulating tumor DNA (ctDNA) for investigation of many cancer types. Using whole-genome next-generation sequencing (NGS), researchers at the Central European Institute of Technology at Masaryk University in the Czech Republic—in collaboration with the EMBL Genomics Core Facility, Heidelberg, Germany—have developed a protocol for efficient cfDNA library preparation that captures short DNA fragments from low amounts of sample to observe copy number variation (CNV), even with low sequencing coverage, using the Collibri PS DNA Library Prep Kit for Illumina Systems.
Video: Success with cfDNA and ctDNA for investigating leukemia activity
Preparation of sequencing-ready libraries from cfDNA or ctDNA presents a number of technical challenges. Despite cfDNA being present in higher levels in cancer patients than in healthy individuals, the total amount of material available is still in the range of nanograms per milliliter of plasma [1]. This issue is further compounded by the low quality of the input material. cfDNA is degraded from its natural origin, generating short fragments with an average of 170 bp. The combination of low input material and intrinsic fragmentation make efficient and reproducible library preparation essential to the performance of liquid biopsies in medical research.
The goal is to recover the shorter, fragmented DNA for downstream analysis using NGS. A customized protocol for efficient library preparation using the Collibri PS DNA Library Prep Kit for Illumina Systems is now available for use with cfDNA or ctDNA samples. The customized protocol for whole-genome sequencing (WGS) permitted Dr. Plevova and the team at EMBL to research disease-specific genomic abnormalities.
Application Note: Library preparation for circulating cfDNA
Modifications to the standard Collibri PS DNA Library Prep Kit for Illumina systems retain short fragments (~170 bp), even from degraded samples. The technical note details how to adapt the standard protocol for use with cfDNA or ctDNA.
Whole-genome library preparation workflow
Genomic DNA extraction
We offer a range of Invitrogen genomic DNA extraction kits for sensitive, scalable purification from an expansive set of starting materials to help maximize process efficiency and downstream performance. This includes a broad range of kits for purifying genomic DNA from a variety of samples including tissue, cells, blood, serum, plants, forensic samples, and more.
Automate DNA extraction using magnetic bead-based technology and KingFisher instruments to bind, wash, and elute DNA to reduce your hands-on time while maintaining high yields and excellent reproducibility.
Sensitive and specific nucleic acid quantification
The Qubit dsDNA high-sensitivity (HS) and broad-range (BR) assays are highly selective for double-stranded DNA (dsDNA) over RNA and single-stranded DNA (ssDNA). The high-sensitivity assay is recommended for samples with a low concentration of dsDNA, making it ideal for precious samples.
Recommended accessories
- Interchange the 96-well 0.2-mL block with another block type if your throughput needs change
- Access the system remotely (and conveniently) through a mobile app
- Program the instrument in seconds with a simple-to-use touchscreen interface
- VeriFlex blocks allow for precise PCR optimization or to run up to six independent assays
- Easy-to-use graphical interface (6.5-inch VGA Touch Screen)
- Reduced PCR cycling time when using faster ramp rates
Quantification options to scale throughput
The qPCR-based Collibri Library Quantification kit scales well for larger sample batches and is the ideal method for precious samples or clinical samples. Quantification accuracy is equivalent to the KAPA Library Quantification kit with the additional benefit of visual feedback during the quantification process.
The Qubit dsDNA HS Assay is a fluorometric assay that uses dsDNA-binding dyes in order to accurately determine NGS library concentration, and benefits from a simple workflow of just a few minutes per sample.
Regardless of the assay that is chosen, good laboratory technique should be used in order to ensure accurate measurement of library concentrations and high-quality Illumina sequencing data.
Recommended accessories
With Applied Biosystems QuantStudio real-time PCR systems, you get true value with excellent performance, reliability, and world-class support. Our family of instruments enables you to obtain the results you need, connect and collaborate with colleagues, and achieve your research goals.
Genomic DNA extraction
We offer a range of Invitrogen genomic DNA extraction kits for sensitive, scalable purification from an expansive set of starting materials to help maximize process efficiency and downstream performance. This includes a broad range of kits for purifying genomic DNA from a variety of samples including tissue, cells, blood, serum, plants, forensic samples, and more.
Automate DNA extraction using magnetic bead-based technology and KingFisher instruments to bind, wash, and elute DNA to reduce your hands-on time while maintaining high yields and excellent reproducibility.
Sensitive and specific nucleic acid quantification
The Qubit dsDNA high-sensitivity (HS) and broad-range (BR) assays are highly selective for double-stranded DNA (dsDNA) over RNA and single-stranded DNA (ssDNA). The high-sensitivity assay is recommended for samples with a low concentration of dsDNA, making it ideal for precious samples.
Recommended accessories
- Interchange the 96-well 0.2-mL block with another block type if your throughput needs change
- Access the system remotely (and conveniently) through a mobile app
- Program the instrument in seconds with a simple-to-use touchscreen interface
- VeriFlex blocks allow for precise PCR optimization or to run up to six independent assays
- Easy-to-use graphical interface (6.5-inch VGA Touch Screen)
- Reduced PCR cycling time when using faster ramp rates
Quantification options to scale throughput
The qPCR-based Collibri Library Quantification kit scales well for larger sample batches and is the ideal method for precious samples or clinical samples. Quantification accuracy is equivalent to the KAPA Library Quantification kit with the additional benefit of visual feedback during the quantification process.
The Qubit dsDNA HS Assay is a fluorometric assay that uses dsDNA-binding dyes in order to accurately determine NGS library concentration, and benefits from a simple workflow of just a few minutes per sample.
Regardless of the assay that is chosen, good laboratory technique should be used in order to ensure accurate measurement of library concentrations and high-quality Illumina sequencing data.
Recommended accessories
With Applied Biosystems QuantStudio real-time PCR systems, you get true value with excellent performance, reliability, and world-class support. Our family of instruments enables you to obtain the results you need, connect and collaborate with colleagues, and achieve your research goals.
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All sequencing methods
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RNA sequencing
Technical notes
For Research Use Only. Not for use in diagnostic procedures.