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Minimal residual disease (MRD) research

Tomorrow’s cancer research breakthroughs are on the horizon. The xGen MRD solution offers a complete sample preparation workflow including custom MRD hybridization capture panels delivered quickly and affordably.

xGen™ NGS—made for cancer research.

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Overview

  • More complete workflow—curated workflow for custom MRD panels
  • Turnaround time—proven TAT of 5 business days for custom capture panels
  • Flexible panel size—target mutations using up to 2,000 probes per panel
  • Affordable MRD research—xGen MRD Custom Hybridization Panel delivers a custom solution for under $40 USD per sample
  • Rare variants—identify variants at ≤1% variant allele frequency (VAF) with your custom panel
  • Automation friendly—easy workflow that can be automated for high-throughput applications (pulled from exome hyb product page for hyb cap automation)
  • PCR analysis options—oPools™ Oligo Pools accommodate multiplexed PCR workflows for MRD research

Minimal residual disease (MRD) refers to the presence of cancerous cells at low levels [1]. MRD research using NGS requires a full solution that encompasses initial variant discovery followed by custom target enrichment of those mutations from low-abundance cell-free DNA (cfDNA). IDT offers an MRD workflow that utilizes the xGen Exome Hyb Panel v2, designed using a target-aware algorithm that reduces off-target binding while maximizing coverage for identifying variants in the original tumor. Those mutations then form the basis for a customized xGen MRD Custom Hybridization Research Panel to be designed, synthesized, and shipped within 5 business days of ordering. With the xGen cfDNA & FFPE DNA Library Preparation Kit v2 Kit, which empowers variant identification from low-input cfDNA research samples, low-level MRD-specific mutations can be investigated using the customized MRD Panel.

As an alternative, IDT offers oPools Oligo Pools that support multiplex PCR workflows for MRD research. oPools Oligo Pools include up 20,000 pooled oligonucleotides that can be from 40–350 bases in length.

Method data

Variant discovery with xGen Exome Hyb Panel v2

Whole exome sequencing (WES) is a targeted next generation sequencing (NGS) approach that uses modified oligonucleotide probes to “capture” and enrich the DNA sample for protein coding regions (exons). Focusing only on protein-coding genomic regions can lower the cost and time of sequencing, as exons make up approximately 1% of the genome but contain 85% of the variants that are associated with disease [2].

The xGen Exome Hyb Panel v2 consists of 415,115 probes that spans a 34 Mb target region (19,433 genes) of the human genome and 39 Mb of probe space—the genomic regions covered by probes. Our probes are designed using a new “capture-aware” algorithm and assessed with proprietary off-target analysis. All probes in the panel are manufactured under ISO13485:2016 standards, and then, mass spectrometry and dual quantification measurements of each probe are performed before they are pooled into the xGen Exome Hyb Panel v2. These measures ensure the quality of the probe and its appropriate representation in the final panel.

On-target coverage and uniformity of the xGen Exome Hyb Panel v2

To test the xGen Exome Hyb Panel v2, a singleplex and 12-plex capture of genomic DNA was done and the resulting fragments were sequenced to assess what percentage of reads were on-target (Figure 1). The same libraries were analyzed for uniformity (Figure 2). Approximately 95% of the reads were on-target for both singleplex and 12-plex, and greater than 97% of the target space had 20X coverage. Three other vendors were compared to the IDT xGen Exome Hyb Panel v2 for on-target coverage, and even though the panels were used according to their recommendations, the on-target percentage was lower than the 12-plex capture using the xGen Exome Hyb Panel v2.

Maximize the number of samples per flowcell

To reduce reagent costs, IDT researchers determined the number of samples that could be loaded onto the NovaSeq™ S2 flowcell (Figure 3). Higher on-target percentages for the xGen Exome Hyb Panel v2 reduces the flowcell space lost to off-target reads, and therefore, more than 110 individual samples can be assessed simultaneously, thus increasing output for customers working with large sample numbers.

Custom MRD workflow with xGen MRD Custom Hyb Panels

Comprehensive conversion and error correction enables ultra-low variant identification with cell-free DNA

The unique, single-stranded ligation strategy of the IDT xGen cfDNA & FFPE DNA Library Prep v2 MC Kit and workflow delivers high conversion of input DNA molecules to sequencing data. This high conversion rate is critical for identification of ultra-low frequency variants, which is common in the analysis of cell-free DNA (cfDNA). A higher conversion rate translates to more complexity and coverage than other DNA library prep kits for cfDNA (Figure 4). In addition, the xGen cfDNA & FFPE DNA Library Prep Kit includes adapters that contain unique molecular identifiers (UMIs), which enable bioinformatic error correction. Combining higher complexity and coverage with stringent error correction better enables the identification of ultra-low frequency variants (Table 1).

Table 1. xGen cfDNA & FFPE DNA Library Prep Kit identifies low frequency variants in NGS reference samples.

MutationExpected VAF (%)xGen cfDNA & FFPE DNA Library Prep MC Kit*Library kit A*Library kit B*
EGFR: L858R0.250.13 (3/3)0.21 (3/3)0.21 (3/3)
EGFR: E746-A7500.250.11 (3/3)0.19 (3/3)0.12 (3/3)
EGFR: T790M0.250.29 (3/3)0.36 (3/3)0.12 (3/3)
KRAS: G12D0.320.33 (3/3)0.36 (3/3)0.33 (3/3)
NRAS: Q61K0.320.23 (3/3)0.31 (2/3)0.22 (3/3)
NRAS: A59T0.320.17 (3/3)0.43 (2/3)0.22 (3/3)
PIK3CA:E545K0.320.16 (3/3)0.11 (3/3)0.36 (3/3)

*Libraries were prepared in triplicate from 50 ng input Horizon cfDNA reference standards using the xGen cfDNA &FFPE DNA Library Prep MC Kit in addition to two other commercially available library prep kits. Libraries were then captured with a 180 kb (target space) xGen Custom Hyb Panel targeting seven identified SNPs using the using the xGen Hybridization and Wash v2 Reagents and Beads. Captured libraries were pooled and sequenced on a NextSeq 500 (Illumina), using a high output 300 cycle kit and the manufacturer's protocol. After subsampling to 85M total reads, the average variant allele frequency for each of the targeted mutations was calculated for each library prep kit using VarDict.

Ordering

Tumor naïve MRD research products

Tumor informed MRD research products

xGen MRD Hyb Panels have been optimized for use with the xGen Hybridization and Wash v2 Kit, xGen Universal Blockers, and xGen Library Amplification Primer Mix, which can be ordered as part of the “Order my design” process below.

Create custom designs for xGen MRD Hyb Panels

For designing xGen MRD Hyb Panels, please consult the IDT NGS design team.

Request design consultation

Order your predesigned panels

If you have predesigned probe sequences, upload them using our submission form.

Order my design

Request MRD workflow consultation

For more information on the MRD workflow solutions, please contact IDT sales.

Request sales consultation

xGen MRD Custom Hyb Panels

References

  1. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev. 2016;35(3):347-376. doi:10.1007/s10555-016-9629-x
  2. Choi M, Scholl UI, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proceedings of the National Academy of Sciences. 2009. 106 (45): 19096–19101. Doi: 10.1073/pnas.0910672106

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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