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Accurate Sample Assignment in a Multiplexed, Ultrasensitive, High-Throughput Sequencing Assay for Minimal Residual
Jack Bartram1, Edward Mountjoy2, Tony Brooks3
1Genetics and Genomic Medicine Program, Institute of Child Health, University College London, London, United Kingdom; Department of Haematology, Great Ormond Street Hospital for Children, London, United Kingdom.
High-throughput sequencing (HTS) offers a sensitive and cost-effective method for monitoring minimal residual disease (MRD) in acute lymphoblastic leukemia. Optimized HTS protocols accurately detect MRD down to 1 in 10^6 copies, ensuring reliable clinical application.
Area of Science:
- Molecular Biology
- Oncology
- Bioinformatics
Background:
- Minimal residual disease (MRD) monitoring is crucial for acute lymphoblastic leukemia (ALL) treatment efficacy.
- Current MRD detection methods face limitations in cost and sensitivity.
- High-throughput sequencing (HTS) presents a promising alternative for sensitive and economical MRD detection.
Purpose of the Study:
- To evaluate the accuracy and identify errors in HTS for Ig heavy-chain gene analysis in MRD detection.
- To develop and optimize a multiplexing strategy for HTS-based MRD monitoring on the Illumina MiSeq platform.
- To introduce a novel demultiplexing pipeline for accurate sample assignment and data integrity.
Main Methods:
- Utilized Illumina MiSeq for HTS of rearranged Ig heavy-chain genes.
- Developed a multiplexing strategy incorporating high-purity oligonucleotides and dual indexing.
- Implemented a probability-based demultiplexing pipeline, Error-Aware Demultiplexer.
- Validated HTS-MRD sensitivity using digital PCR-quantified controls.
Main Results:
- Identified multiplexing-associated errors impacting MRD accuracy.
- Optimized strategy significantly minimized errors and maximized sensitivity in HTS-MRD analysis.
- The Error-Aware Demultiplexer pipeline accurately assigned samples with minimal data loss.
- Demonstrated HTS-MRD can detect leukemic MRD at a level of 1 in 10^6 copies.
Conclusions:
- Optimized HTS with dual indexing and error-aware demultiplexing ensures accurate and sensitive MRD detection in ALL.
- The developed pipeline is suitable for MiSeq-based HTS strategies, enhancing clinical laboratory adoption.
- HTS-MRD is a viable, highly sensitive tool for monitoring minimal residual disease in leukemia patients.
