Illumina TruSight Oncology 500: VWR Deep Well Plate vs. Fisher Scientific MIDI Plate Assessment
Document ID: M-GL-03612 v1.0
For Research Use Only. Not for use in diagnostic procedures.
Introduction
Illumina TruSight Oncology 500 is a comprehensive next-generation sequencing (NGS) assay for comprehensive genomic profiling (CGP). Due to ongoing shortages of certain plastic consumables, laboratories have faced difficulties sourcing the Fisher Scientific 96-well 0.8 ml MIDI plate (Thermo Fisher Scientific, Catalog no. AB-0859), a consumable specified in the TruSight Oncology 500 Reference Guide. This technical note reviews the use of the VWR 96-well Deep Well Plate (VWR, Catalog no. 76210-524) as an alternative in the library preparation steps within the TruSight Oncology 500 Research Use Only workflow.
The thermal properties of the Fisher Scientific and VWR plates were compared, and differences in library sequence performance were assessed. While the results indicate that the VWR plate may be a viable substitute for the specified use, this substitution has not been validated by Illumina, and performance is not guaranteed. It is recommended that laboratories independently evaluate and verify performance before adopting this alternative in their workflows.
Methods
Thermal and functional tests were conducted to evaluate the use of VWR 96-well Deep Well Plates as an alternative to Fisher Scientific 96-well MIDI plates in the Illumina TruSight Oncology 500 workflow. All incubations for thermal and functional testing were performed on the same Hybex Microsample Incubator (SciGene, Catalog no. 1057-30-O) with an Illumina MIDI Heat Block Insert (Illumina, Catalog no. BD-60-601).
Fisher Scientific is a brand of Thermo Fisher Scientific. VWR is a subsidiary of Avantor.
Plate Specifications
| Supplier | Product Name | Catalog No. | Plastic Type | Volume per well | Plate weight | Sterility | Max centrifuge |
|---|---|---|---|---|---|---|---|
| Thermo Fisher Scientific | Fisher Scientific 96-well MIDI storage platea | AB-0859 | Polypropylene | 0.8 ml | 78.0 g | DNase/ RNase Free | 2000 × g |
| VWR/Avantor | VWR 96-well Deep Well Plate | 76210-524 | Polypropylene | 0.8 ml | 77.0 g | DNase/ RNase Free | 10,000 × g |
a Current full product name is Abgene 96 Well 0.8mL Polypropylene DeepWell Sample Processing & Storage Plate for Genomics and NGS library preparation.
Testing Procedures
Plates were assessed for differences in heating properties by adding 100 µl nuclease-free dH2O to five wells per plate at 57°C for five minutes and recording final temperatures with a thermocouple (Extech, Catalog no. TM500). Three replicates of each plate type were assessed.
To evaluate nonspecific DNA binding, 25 ng of fragmented Seraseq reference control DNA (SeraCare, Catalog no. 10592800) in 100 µl Resuspension Buffer (RSB; included in the TruSight Oncology 500 library prep kit) was incubated at 72°C for 20 minutes. Pre- and post-incubation concentrations were measured using the Qubit 1X dsDNA High Sensitivity (HS) Assay Kit (Thermo Fisher Scientific, Catalog no. Q33230).
DNA and RNA libraries were prepared following the TruSight Oncology 500 standard protocol. Libraries were prepared from 40 ng Seraseq reference control DNA or Seraseq reference control RNA (SeraCare, Catalog no. 10597544) and 40 ng DNA or RNA extracted from FFPE samples (Illumina internal samples). For comparative analysis, libraries were prepared in parallel using either the Fisher Scientific plate or the VWR plate, with each plate type used consistently throughout all steps of the protocol that specified the MIDI plate. Library prep was performed manually or with partial automation using the Hamilton Microlab STAR Liquid Handler (Hamilton, Catalog no. HMLT-STAR-B575), with all manual steps executed by the same operator.
Functional runs consisted of sequencing prepared libraries on the Illumina NextSeq™ 550 System and analysis using the TruSight Oncology Comprehensive Analysis Module V2.9.5. Data were evaluated using run-level and library-level QC metrics, including median insert size, usable microsatellite instability (MSI) sites, percent of reads on target (PCT target), and median exon coverage for DNA samples, and median insert size, total on-target reads, and median CV gene 500K for RNA samples. The FFPE-derived DNA and RNA samples each contained a variant in one of the targeted regions to allow for sensitivity evaluation.
Results
Thermal Properties
Thermal measurements revealed that wells in the VWR plates consistently reached 1–2°C lower final temperatures than wells in the Fisher Scientific plates under identical heating conditions. Nonspecific DNA binding tests showed no significant difference between the Fisher Scientific and VWR plates, with comparable DNA recovery following incubation at 72°C.
Control Sample Performance
Experiments using reference control samples included five functional runs using eight DNA and RNA replicates each. Libraries prepared with VWR plates showed reduced coverage metrics for both DNA and RNA control samples compared to samples prepared with Fisher Scientific plates.
DNA Reference Control (Figure 1A): While DNA libraries still met all specification limits, the VWR plate showed slightly lower performance in metrics like median exon coverage and PCT target. Statistical analysis (t-tests) indicated significant differences for median insert size, usable MSI sites, and median exon coverage, with VWR generally showing lower values.
RNA Reference Control (Figure 1B): RNA libraries showed reduced coverage, elevated median insert sizes, and an increased rate of functional failures due to shifts in total on-target reads. Statistical analysis (t-tests) indicated significant differences for all assessed metrics (median insert size, median CV gene 500K, total on-target reads), with VWR performing worse in these areas.
A subsequent RNA-only run using pooled cDNA libraries confirmed that libraries generated with VWR plates exhibited consistently lower total on-target reads, with approximately a 3% decrease compared to Fisher Scientific plates.
Key Definitions: CV (coefficient of variation), LSL (lower specification limit), MSI (microsatellite instability), PCT (percent target bases), SEM (standard error of the mean), USL (upper specification limit).
FFPE Sample Performance
For functional runs using FFPE-derived samples, eight replicates each of DNA and RNA samples were evaluated. Libraries were treated identically except for steps using the MIDI plates, with half processed using Fisher Scientific plates and the other half using VWR plates.
FFPE DNA (Figure 2A): Libraries prepared from FFPE-derived DNA samples showed equivalent performance between plate types. No observable differences were found in library QC metrics such as median exon coverage, PCT Target 100X, usable MSI sites, or median insert size. Sensitivity for detecting expected variants was maintained across all runs, regardless of plate type.
FFPE RNA (Figure 2B): Libraries prepared from FFPE-derived RNA samples also showed equivalent performance between plate types. No observable differences were found in library QC metrics such as total PF reads, total on-target reads, or median insert size. Sensitivity for detecting expected variants was maintained across all runs, regardless of plate type.
Summary
The VWR 96-well Deep Well Plate performed comparably to the Fisher Scientific 96-well MIDI storage plate for libraries prepared from DNA reference control and FFPE-derived DNA and RNA samples. However, its use was associated with reduced library coverage and increased failure rates when using RNA reference control samples. The VWR plates have been evaluated solely for the TruSight Oncology 500 Research Use Only workflow. It is recommended that laboratories assess the performance of the VWR plate within their own workflows before implementation.
Reference
1. Illumina. TruSight Oncology 500 Reference Guide. Available at: support.illumina.com/documents/documentation/chemistry_documentation/trusight/oncology-500/1000000067621_11_trusight-oncology-500-reference-guide.pdf. Published January 2023. Accessed May 22, 2025.
