Accurate Sample Assignment in a Multiplexed, Ultrasensitive, High-Throughput Sequencing Assay for Minimal Residual Disease

Jack Bartram, Edward Mountjoy, Tony Brooks, Jeremy Hancock, Helen Williamson, Gary Wright, John Moppett, Nick Goulden, Mike Hubank

Research output: Contribution to journalArticle (Academic Journal)peer-review

25 Citations (Scopus)

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 106 copies of specific leukemic MRD.
Original languageEnglish
Pages (from-to)494-506
Number of pages13
JournalJournal of Molecular Diagnostics
Volume18
Issue number4
Early online date13 May 2016
DOIs
Publication statusPublished - Jul 2016

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