Date Published: April 30, 2019
Publisher: Public Library of Science
Author(s): Rebecca E. Colman, Aurélien Mace, Marva Seifert, Jonathan Hetzel, Haifa Mshaiel, Anita Suresh, Darrin Lemmer, David M. Engelthaler, Donald G. Catanzaro, Amanda G. Young, Claudia M. Denkinger, Timothy C. Rodwell, Richard Chaisson
Abstract: BackgroundAccurate, comprehensive, and timely detection of drug-resistant tuberculosis (TB) is essential to inform patient treatment and enable public health surveillance. This is crucial for effective control of TB globally. Whole-genome sequencing (WGS) and targeted next-generation sequencing (NGS) approaches have potential as rapid in vitro diagnostics (IVDs), but the complexity of workflows, interpretation of results, high costs, and vulnerability of instrumentation have been barriers to broad uptake outside of reference laboratories, especially in low- and middle-income countries. A new, solid-state, tabletop sequencing instrument, Illumina iSeq100, has the potential to decentralize NGS for individual patient care.Methods and findingsIn this study, we evaluated WGS and targeted NGS for TB on both the new iSeq100 and the widely used MiSeq (both manufactured by Illumina) and compared sequencing performance, costs, and usability. We utilized DNA libraries produced from Mycobacterium tuberculosis clinical isolates for the evaluation. We conducted WGS on three strains and observed equivalent uniform genome coverage with both platforms and found the depth of coverage obtained was consistent with the expected data output. Utilizing the standardized, cloud-based ReSeqTB bioinformatics pipeline for variant analysis, we found the two platforms to have 94.0% (CI 93.1%–94.8%) agreement, in comparison to 97.6% (CI 97%–98.1%) agreement for the same libraries on two MiSeq instruments. For the targeted NGS approach, 46 M. tuberculosis–specific amplicon libraries had 99.6% (CI 98.0%–99.9%) agreement between the iSeq100 and MiSeq data sets in drug resistance–associated SNPs. The upfront capital costs are almost 5-fold lower for the iSeq100 ($19,900 USD) platform in comparison to the MiSeq ($99,000 USD); however, because of difference in the batching capabilities, the price per sample for WGS was higher on the iSeq100. For WGS of M. tuberculosis at the minimum depth of coverage of 30x, the cost per sample on the iSeq100 was $69.44 USD versus $28.21 USD on the MiSeq, assuming a 2 × 150 bp run on a v3 kit. In terms of ease of use, the sequencing workflow of iSeq100 has been optimized to only require 27 minutes total of hands-on time pre- and post-run, and the maintenance is simplified by a single-use cartridge–based fluidic system. As these are the first sequencing attempts on the iSeq100 for M. tuberculosis, the sequencing pool loading concentration still needs optimization, which will affect sequencing error and depth of coverage. Additionally, the costs are based on current equipment and reagent costs, which are subject to change.ConclusionsThe iSeq100 instrument is capable of running existing TB WGS and targeted NGS library preparations with comparable accuracy to the MiSeq. The iSeq100 has reduced sequencing workflow hands-on time and is able to deliver sequencing results in <24 hours. Reduced capital and maintenance costs and lower-throughput capabilities also give the iSeq100 an advantage over MiSeq in settings of individualized care but not in high-throughput settings such as reference laboratories, where sample batching can be optimized to minimize cost at the expense of workflow complexity and time.
Partial Text: Tuberculosis (TB) mortality has been declining at a rate of approximately 3% per year since 2000 . Although encouraging, this trajectory will not lead to achieving the End TB target of a 95% reduction of TB deaths by 2035 . Given that drug-resistant TB (DR-TB) is a key driving factor behind TB mortality globally, implementation of comprehensive, rapid drug susceptibility testing (DST) is critical in all settings. Detection of resistance-conferring mutations by molecular methods is a rapid and accurate alternative to phenotypic DST and has been shown to provide actionable information to healthcare workers (e.g., GeneXpert MTB/RIF) . Currently, molecular resistance tests provide only partial data on drug susceptibility and are limited by the number of gene targets examined. With the introduction of novel and repurposed drugs, new treatment regimens, and increasing numbers of patients with complex resistance profiles, a more comprehensive solution for guiding patient treatment becomes crucial. The rapid evolution of knowledge on the genetic determinants of TB drug resistance suggests that gene sequencing will become the most appropriate and versatile technology platform to provide rapid, accurate, and actionable results for treatment of this disease [3,4]. A next-generation sequencing (NGS) approach gives comprehensive genetic information on drug resistance–related genes, be it through whole-genome sequencing (WGS) or targeted NGS .
Adoption of NGS to examine a multitude of questions in TB research has rapidly occurred over the past few years. The use of NGS for comprehensive drug resistance profiles through examination of drug resistance–associated mutations is a promising tool for clinical care. Moving NGS workflows into clinical settings from research settings will require an evaluation of the performance of established workflows on different sequencing instruments, possibly opening new avenues for establishing NGS for TB clinical use. In this study, the iSeq100 platform’s performance was comparable to the commonly used MiSeq platform (Fig 1, S1 Table), illustrating the potential interchangeability of different sequencers in a laboratory setting.