Illumina TruSight Oncology 500 Automated on the Biomek NXᴾ Span-8 Genomics Workstation

Introduction

The TruSight Oncology 500 assay is a comprehensive next-generation sequencing (NGS) assay targeting the full coding regions of 523 genes implicated in the pathogenesis of solid tumors. Using hybrid-capture based library preparation techniques for use with formalin-fixed, paraffin-embedded (FFPE tissue) samples, TruSight Oncology 500 can analyze DNA and RNA from the same sample, detecting single nucleotide variants (SNVs), indels, amplifications, splice variants, and fusions, in a single sequencing run. TruSight Oncology 500 also allows for the determination of tumor mutational burden (TMB) and microsatellite instability (MSI) in DNA samples, which are currently being developed as biomarkers for immunotherapy. There are two library preparation kit options for the TruSight Oncology 500 assay depending on sample type: PN 20028213 (DNA kit only) or PN 20028215 (DNA and RNA kit bundle). The Biomek automated TruSight Oncology 500 workflow guarantees 32 library reactions can be prepared from the library preparation kit. If running DNA only samples, the automated Illumina TruSight Oncology 500 workflow PN 20028213 (DNA kit only) supports 3 to 32 DNA libraries in one run. If running RNA (derived from FFPE or cell lines) and DNA (derived from FFPE or cell lines) simultaneously, the automated Illumina TruSight Oncology 500 workflow PN 20028215 (DNA and RNA kit bundle) supports a combination of RNA and DNA libraries up to a total of 32 libraries. The maximum number of RNA libraries that can be processed in one run is 16. If running less than 16 RNA libraries, additional DNA libraries can be processed in one run (up to the maximum total of 32 libraries per run).

In this flyer, we demonstrate the automated performance of Illumina TruSight Oncology 500 assay on the Biomek NXP Span-8 Genomics Workstation.

In comparison to the manual pipetting, automating the Illumina TruSight Oncology 500 assay on Biomek platforms provides

  • Reduced hands-on time
  • Reduction in potential pipetting errors
  • Quick implementation with ready-to-implement methods
  • Knowledgeable support

Spotlight

Biomek NXP Span-8 Genomics Workstation features deliver reliability and efficiency to increase user confidence and walk-away time

  • Span-8 pod with disposable tips capable of transfers ranging from 1 µL to 1000 µL
  • Independent 360O rotating gripper
  • Orbital Shaker, peltiers, and liquid waste disposal for sample processing

Figure 1. Biomek NXP Span-8 Genomics Workstation

Figure 1. Biomek NXP Span-8 Genomics Workstation

Automated Method

The automated TruSight Oncology 500 method is constructed in a modular fashion that follows the manual assay’s recommended start and stop points, allowing the operator flexibility in performing the assay and allowing the automation method to be easily deployed in pre-PCR and post-PCR laboratory spaces. Hybridization reactions and PCR reactions are performed on off-deck thermocyclers to allow for more efficient instrument utilization, but all other incubations are performed on-deck.

Figure 2. Illumina TruSight Oncology 500 automated method workflow

Figure 2. Illumina TruSight Oncology 500 automated method workflow

Automation provides increased efficiency, reducing the hands-on time (Tables 1 and 2). 

8 DNA and 8 RNA Samples
Day 1
Time
Biomek Setup Biomek Runtime Manual procedure Non-Biomek Instrument Time
Denature and Anneal RNA, First Strand Synthesis, Second Strand cDNA Synthesis, and cDNA Cleanup 15 min 2 hr, 16 min
Covaris Shearing 15 min 2 hr
End Repair and A-Tailing, Ligate Adapters, and Clean 15 min 2 hr, 35 min
Indexing PCR 10 min 9 min 40 min
First Hybridization 10 min 12 min Over Night
Totals 50 min 5 hr, 12 min 15 min >18 hr

Day 2
Time
Biomek Setup Biomek Runtime Manual procedure Non-Biomek Instrument Time
First Capture 15 min 1 hr, 44 min
Second Hybridization 10 min 11 min 1 hr, 30 min
Second Capture 15 min 1 hr, 8 min
Amplify Enriched Libraries 9 min 50 min
Clean Up Amplified Libraries 15 min 37 min
Library Quantification 20 min
Normalize Libraries 15 min 1 hr, 12 min
Sequencing Setup 20 min
Totals 1 hr, 10 min 5 hr, 1 min 40 min 2 hr, 20 min

Table 1. Estimated run times for processing 8 RNA and 8 DNA samples on the automated Illumina TruSight Oncology 500 method

 

32 DNA Samples
Day 1
Time
Biomek Setup Biomek Runtime Manual procedure Non-Biomek Instrument Time
Denature and Anneal RNA, First Strand Synthesis, Second Strand cDNA Synthesis, and cDNA Cleanup
Covaris Shearing 15 min 2 hr
End Repair and A-Tailing, Ligate Adapters, and Clean 15 min 2 hr, 56 min
Indexing PCR 10 min 12 min 40 min
First Hybridization 10 min 20 min Over Night
Totals 35 min 3 hr, 28 min 15 min >18 hr

Day 2
Time
Biomek Setup Biomek Runtime Manual procedure Non-Biomek Instrument Time
First Capture 15 min 2 hr, 2 min
Second Hybridization 10 min 18 min 1 hr, 30 min
Second Capture 15 min 1 hr, 18 min
Amplify Enriched Libraries 10 min 50 min
Clean Up Amplified Libraries 15 min 49 min
Library Quantification 20 min
Normalize Libraries 15 min 1 hr, 21 min
Sequencing Setup 25 min
Totals 1 hr, 10 min 5 hr, 58 min 40 min 2 hr, 20 min

Table 2. Estimated run times for processing 32 DNA samples on the automated Illumina TruSight Oncology 500 method

The software provides several user-friendly features such as

1. Biomek Method Launcher (BML)

BML is a secure interface for method implementation without affecting method integrity. It allows the users to remotely monitor the progress of the run. The manual control options provide the opportunity to interact with the instrument, if needed. The application can also be run through standard Biomek software if Biomek Method Launcher is not available.

Figure 3. Biomek Method Launcher provides an easy interface to launch the method.

Figure 3. Biomek Method Launcher provides an easy interface to launch the method.

2. Method Options Selector (MOS)

MOS enables selection of plate processing and sample number options to maximize flexibility, adaptability and the ease of method execution

Figure 4. Biomek Method Options Selector enables us to select the desired workflow, sample number, and a variety of workflow customization options

Figure 4. Biomek Method Options Selector enables us to select the desired workflow, sample number, and a variety of workflow customization options

3. Guided Labware Setup (GLS)

GLS is generated based on options selected in the MOS, and provides the user specific graphical setup instructions with reagent volume calculation and step by step instructions to prepare reagents.

Figure 5. Guided Labware Setup indicates reagent volumes and guides the user for correct deck setup

Figure 5. Guided Labware Setup indicates reagent volumes and guides the user for correct deck setup

Experiment Design

Using two Biomek NXP Span-8 systems, a reproducibility study was performed consisting of three runs of the Illumina TruSight Oncology 500 automated method on each Biomek NXP Span-8 along
with manual controls. For each manual and automated run, 8 RNA and 24 wells of DNA samples were processed. RNA samples included four SeraCare RNA replicates (Seraseq Tumor Fusion RNA Mix v3 Part # 0710-0431) and and four FFPE RNA samples from Illumina. DNA samples included four SeraCare DNA replicates (Seraseq Tri-Lvl Mut DNA Mix v2 HC Part # 0710-0097) and 20 FFPE DNA samples from Illumina with ΔCq values ranging from 1.83 to 4.11. After library construction, sequencing and analysis was performed at Illumina. A subset of the DNA FFPE sample and cell line sample replicates are shown below, with the SeraCare DNA replicates identified as “DNA Cell Line Mix” in the following figures.

Figure 6. Percent exon coverage at 50X by sample.

Figure 6. Percent exon coverage at 50X by sample.

Figure 7. Total TMB (Tumor Mutation Burden) score by sample.

Figure 7. Total TMB (Tumor Mutation Burden) score by sample.

Figure 8. Percent MSI (Microsatellite Instability) sites by sample.

Figure 8. Percent MSI (Microsatellite Instability) sites by sample.

RNA libraries prepared on the Biomek NXP Span-8 system performed comparable to bench and passed all QC thresholds set by Illumina as shown in Table 3.

Platform Libraries Passed AccuClear Median Insert Size Median_CV_GENE_500X Total_On_Target_Reads
Manual Assay (N=24) 92% 100% 100% 92%
Automated NXP Assay (N=48) 98% 100% 98% 98%

Table 3. RNA library metrics

Summary

A robust and reliable automation solution for NGS library prep is essential to take full advantage of Illumina’s sequencing technology. Automation of the library preparation process can offer laboratories significant time savings compared to manual preparation. For example, manual user is estimated to take approximately 6 hours and 30 minutes of hands-on time to prepare 8 RNA and 8 DNA libraries, while the Biomek NXP Span-8 Genomics Workstation can prepare same number of libraries with less than 3 hours of hands-on time.

We’ve demonstrated automation of the Illumina TruSight Oncology 500 assay on the Biomek NXP Span-8 Genomics Workstation delivers libraries that yield quality results across sample types of varying quality.

 

The TruSight Oncology 500 library preparation kit is for Research Use Only. The TruSight Oncology 500 library preparation kit is not for use in diagnostic procedures. The Biomek Workstation is not intended or validated for use in the diagnosis of disease or other conditions. Beckman Coulter makes no warranties of any kind whatsoever, express or implied, with respect to this protocol, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated for use in the diagnosis of disease or other conditions. This protocol is for demonstration only and is not validated by Beckman Coulter.

Biomek Method Launcher software package must be purchased separately.

 

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