title: Accurate Sample Assignment in a Multiplexed, Ultrasensitive, High-Throughput Sequencing Assay for Minimal Residual Disease.
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.
authors.affiliation: Genetics 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.
authors.forename: Jack
authors.initials: J
authors.lastname: Bartram
authors.affiliation: School of Social and Community Medicine, University of Bristol, Bristol, United Kingdom.
authors.forename: Edward
authors.initials: E
authors.lastname: Mountjoy
authors.affiliation: UCL Genomics Centre, Institute of Child Health, University College London, London, United Kingdom.
authors.forename: Tony
authors.initials: T
authors.lastname: Brooks
authors.affiliation: Bristol Genetics Laboratory, Southmead Hospital, North Bristol NHS Trust, Bristol, United Kingdom.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Hancock
authors.affiliation: Bristol Genetics Laboratory, Southmead Hospital, North Bristol NHS Trust, Bristol, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Williamson
authors.affiliation: Department of Haematology, Great Ormond Street Hospital for Children, London, United Kingdom.
authors.forename: Gary
authors.initials: G
authors.lastname: Wright
authors.affiliation: Department of Paediatric Haematology/Oncology, Royal Hospital for Children, Bristol, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Moppett
authors.affiliation: Department of Haematology, Great Ormond Street Hospital for Children, London, United Kingdom.
authors.forename: Nick
authors.initials: N
authors.lastname: Goulden
authors.affiliation: Genetics and Genomic Medicine Program, Institute of Child Health, University College London, London, United Kingdom; UCL Genomics Centre, Institute of Child Health, University College London, London, United Kingdom. Electronic address: michael.hubank@icr.ac.uk.
authors.forename: Mike
authors.initials: M
authors.lastname: Hubank
mesh_terms: D019295:Computational Biology / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012984:Software