eprintid: 1501080 rev_number: 34 eprint_status: archive userid: 608 dir: disk0/01/50/10/80 datestamp: 2016-06-23 10:27:41 lastmod: 2022-01-10 00:20:54 status_changed: 2016-06-23 10:27:41 type: article metadata_visibility: show creators_name: Bartram, J creators_name: Mountjoy, E creators_name: Brooks, T creators_name: Hancock, J creators_name: Williamson, H creators_name: Wright, G creators_name: Moppett, J creators_name: Goulden, N creators_name: Hubank, M title: Accurate sample assignment in a multiplexed, ultrasensitive, high-throughput sequencing assay for minimal residual disease ispublished: pub divisions: UCL divisions: B02 divisions: D13 note: © 2016 Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology and the Association for Molecular Pathology. All rights reserved. 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. date: 2016-05-13 date_type: published official_url: http://dx.doi.org/10.1016/j.jmoldx.2016.02.008 oa_status: green full_text_type: other language: eng primo: open primo_central: open_green article_type_text: JOURNAL ARTICLE verified: verified_manual elements_id: 1137809 doi: 10.1016/j.jmoldx.2016.02.008 pii: S1525-1578(16)30045-9 language_elements: ENG lyricists_name: Bartram, Jack lyricists_name: Hubank, Michael lyricists_id: JLBAR39 lyricists_id: MJFHU53 actors_name: Bartram, Jack actors_name: Poirier, Elizabeth actors_id: JLBAR39 actors_id: EPPOI23 actors_role: owner actors_role: impersonator full_text_status: public publication: Journal of Molecular Diagnostics volume: 18 number: 4 pagerange: 494-506 issn: 1943-7811 citation: Bartram, J; Mountjoy, E; Brooks, T; Hancock, J; Williamson, H; Wright, G; Moppett, J; ... Hubank, M; + view all <#> Bartram, J; Mountjoy, E; Brooks, T; Hancock, J; Williamson, H; Wright, G; Moppett, J; Goulden, N; Hubank, M; - view fewer <#> (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 <https://doi.org/10.1016/j.jmoldx.2016.02.008>. Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/1501080/1/Bartram_JMolDiagnostics_final.pdf