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Workflow optimization of whole genome amplification and targeted panel sequencing for CTC mutation detection
Haiyan E Liu1, Melanie Triboulet2, Amin Zia3
1Vortex Biosciences, Inc., Menlo Park, CA USA.
NPJ Genomic Medicine
|December 22, 2017
Summary
Genomic analysis of circulating tumor cells (CTCs) offers a non-invasive alternative to biopsies. A new workflow using Vortex enrichment and REPLI-g whole genome amplification (WGA) enables accurate detection of tumor mutations in CTCs.
Area of Science:
- Oncology
- Genomics
- Biotechnology
Background:
- Circulating tumor cells (CTCs) offer a minimally invasive method for cancer monitoring.
- Challenges exist in isolating sufficient DNA from rare CTCs for genomic analysis.
- Germline DNA from white blood cells (WBCs) can contaminate CTC samples, complicating mutation detection.
Purpose of the Study:
- To develop and validate a workflow for genomic characterization of CTCs.
- To evaluate label-free CTC enrichment, whole genome amplification (WGA), and targeted next-generation sequencing (NGS).
- To assess the feasibility of real-time monitoring of tumor genetics using CTCs in metastatic colorectal cancer (CRC).
Main Methods:
- Vortex platform for label-free CTC enrichment.
- Optimization of DNA extraction and evaluation of WGA methods (GenomePlex®, REPLI-g).
- Targeted NGS using a 38-gene CRC panel, validated on cell lines and patient samples, including WBCs for contamination control.
Main Results:
- The workflow demonstrated effectiveness with sufficient DNA input.
- REPLI-g WGA showed unbiased amplification from rare cells with limited DNA.
- Accurate variant calling was achieved using targeted NGS, detecting somatic variants in patient CTCs not present in healthy donors.
Conclusions:
- A simplified workflow for CTC genomic analysis is feasible.
- REPLI-g WGA is suitable for unbiased amplification of rare cell DNA.
- This approach enables real-time monitoring of tumor genetics during therapy via CTCs.
