Bartram, J;
Mountjoy, E;
Brooks, T;
Hancock, J;
Williamson, H;
Wright, G;
Moppett, J;
... Hubank, M; + view all
(2016)
Accurate sample assignment in a multiplexed, ultrasensitive, high-throughput sequencing assay for minimal residual disease.
Journal of Molecular Diagnostics
, 18
(4)
pp. 494-506.
10.1016/j.jmoldx.2016.02.008.
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Abstract
High-throughput sequencing (HTS) (next-generation sequencing) of the rearranged Ig and T-cell receptor genes promises to be less expensive and more sensitive than current methods of monitoring minimal residual disease (MRD) in patients with acute lymphoblastic leukemia. However, the adoption of new approaches by clinical laboratories requires careful evaluation of all potential sources of error and the development of strategies to ensure the highest accuracy. Timely and efficient clinical use of HTS platforms will depend on combining multiple samples (multiplexing) in each sequencing run. Here we examine the Ig heavy-chain gene HTS on the Illumina MiSeq platform for MRD. We identify errors associated with multiplexing that could potentially impact the accuracy of MRD analysis. We optimize a strategy that combines high-purity, sequence-optimized oligonucleotides, dual indexing, and an error-aware demultiplexing approach to minimize errors and maximize sensitivity. We present a probability-based, demultiplexing pipeline Error-Aware Demultiplexer that is suitable for all MiSeq strategies and accurately assigns samples to the correct identifier without excessive loss of data. Finally, using controls quantified by digital PCR, we show that HTS-MRD can accurately detect as few as 1 in 10(6) copies of specific leukemic MRD.
| Type: | Article |
|---|---|
| Title: | Accurate sample assignment in a multiplexed, ultrasensitive, high-throughput sequencing assay for minimal residual disease |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.jmoldx.2016.02.008 |
| Publisher version: | http://dx.doi.org/10.1016/j.jmoldx.2016.02.008 |
| Language: | English |
| Additional information: | © 2016 Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology and the Association for Molecular Pathology. All rights reserved. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1501080 |
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