Versatile ion S5XL sequencer for targeted next generation sequencing of solid tumors in a clinical laboratory

Meenakshi Mehrotra, Dzifa Yawa Duose, Rajesh R Singh, Bedia A Barkoh, Jawad Manekia, Michael A Harmon, Keyur P Patel, Mark J Routbort, L Jeffrey Medeiros, Ignacio I Wistuba, Rajyalakshmi Luthra, Meenakshi Mehrotra, Dzifa Yawa Duose, Rajesh R Singh, Bedia A Barkoh, Jawad Manekia, Michael A Harmon, Keyur P Patel, Mark J Routbort, L Jeffrey Medeiros, Ignacio I Wistuba, Rajyalakshmi Luthra

Abstract

Background: Next generation sequencing based tumor tissue genotyping involves complex workflow and a relatively longer turnaround time. Semiconductor based next generation platforms varied from low throughput Ion PGM to high throughput Ion Proton and Ion S5XL sequencer. In this study, we compared Ion PGM and Ion Proton, with a new Ion S5XL NGS system for workflow scalability, analytical sensitivity and specificity, turnaround time and sequencing performance in a clinical laboratory.

Methods: Eighteen solid tumor samples positive for various mutations as detected previously by Ion PGM and Ion Proton were selected for study. Libraries were prepared using DNA (range10-40ng) from micro-dissected formalin-fixed, paraffin-embedded (FFPE) specimens using the Ion Ampliseq Library Kit 2.0 for comprehensive cancer (CCP), oncomine comprehensive cancer (OCP) and cancer hotspot panel v2 (CHPv2) panel as per manufacturer's instructions. The CHPv2 were sequenced using Ion PGM whereas CCP and OCP were sequenced using Ion Proton respectively. All the three libraries were further sequenced individually (S540) or multiplexed (S530) using Ion S5XL. For S5XL, Ion chef was used to automate template preparation, enrichment of ion spheres and chip loading. Data analysis was performed using Torrent Suite 4.6 software on board S5XL and Ion Reporter. A limit of detection and reproducibility studies was performed using serially diluted DLD1 cell line.

Results: A total of 241 variant calls (235 single nucleotide variants and 6 indels) expected in the studied cohort were successfully detected by S5XL with 100% and 97% concordance with Ion PGM and Proton, respectively. Sequencing run time was reduced from 4.5 to 2.5 hours with output range of 3-5 GB (S530) and 8-9.3Gb (S540). Data analysis time for the Ion S5XL is faster 1 h (S520), 2.5 h (S530) and 5 h (S540) chip, respectively as compared to the Ion PGM (3.5-5 h) and Ion Proton (8h). A limit detection of 5% allelic frequency was established along with high inter-run reproducibility.

Conclusion: Ion S5XL system simplified workflow in a clinical laboratory, was feasible for running smaller and larger panels on the same instrument, had a shorter turnaround time, and showed good concordance for variant calls with similar sensitivity and reproducibility as the Ion PGM and Proton.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. NGS workflow used for different…
Fig 1. NGS workflow used for different Ion Torrent platforms in a clinical laboratory.
Ion PGM, Ion Proton and Ion S5XL sequencing workflow using cancer hot spot panel v2 (CHPv2), oncomine panel (OCP) and comprehensive cancer panel (CCP) for library prep. Ion one touch 2 and Ion one touch ES was used for PGM and Proton for template preparation and enrichment of ion spheres. Enriched ion spheres were sequenced on Ion PGM and Ion Proton respectively. For Ion S5XL Ion chef was used which automates the template preparation, enrichment and chip loading. Preloaded reagent cartridge and on board Torrent suite software reduces the initialization, sequencing and data analysis time.
Fig 2. Variant call comparison between Ion…
Fig 2. Variant call comparison between Ion Proton and Ion S5XL.
(A) Venn diagram showed variant call comparison between Ion Proton and Ion S5Xl Ion S5XL showed 97% concordance for mutation detection with Ion Proton. Eight variant calls were missed by Ion S5XL due to low coverage in the variant region of the CCP panel compared to Ion Proton (B) Correlation between Variant allelic Fraction (VAF) of mutations detected on protons and Ion S5 XL (R2 = 0.70; p<0.0001) and each point on the graph represents a single variant analyzed by the Ion Proton and S5XL platforms.
Fig 3. Limit of detection study performed…
Fig 3. Limit of detection study performed on Ion S5 XL platform using S530 chip.
Sensitivity study using DLD1 cell line serially diluted in wild type HL60 cell line DNA at 50, 25, 12.5, 6.25, 3.12, 1.50, and 0.75%. DLD1 cell line harbors heterozygous mutations in six different genes: PIK3CA, KRAS, KIT, TP53, FGFR1 and SMO.
Fig 4. Inter-reproducibility studies performed on Ion…
Fig 4. Inter-reproducibility studies performed on Ion S5 XL platform using S530 chip.
Reproducibility studies using one normal tumor paired CCP libraries and one OCP libraries across two different sequencing runs on S5XL platform.

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