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xGen™ HS EGFR Pathway Amplicon Panel

Targeted sequencing panel for EGFR, BRAF, KRAS, and NRAS

xGen HS EGFR Pathway Amplicon Panel can detect variants with a MAF of ≤1% from 10—20 ng of cell-free DNA (cfDNA) or FFPE DNA for Illumina® sequencers.

xGen NGS—made sensitive.

Ordering

  • Identifies SNVs and indels down to 0.25% allele frequency
  • Compatible with cfDNA and FFPE DNA
  • Amplifies from 10–50 ng of cfDNA
  • Makes Illumina®-compatible libraries within 3 hours
  • Supports research in oncology, graft vs. host disease, and single nucleotide variants in the EGFR pathway

Product details

The IDT xGen HS EGFR Pathway Amplicon Panel covers contiguous regions of the EGFR gene, and hotspot coverage of BRAF, KRAS, and NRAS with a panel of amplicon-generating primers. The final amplicons average only 136 bp making them compatible with DNA fragments from cell-free DNA (cfDNA) and/or FFPE DNA. The final target size of this panel is 1.5 kbp.

As seen in Figure 1, this workflow includes three PCR steps-one for the incorporation of molecular identifiers (MIDs) (PCR I), one for target amplification (PCR II), and one for the addition of combinatorial dual indexed adapters (PCR III), enabling multiplexing of up to 96 unique libraries. Bead-based SPRI® cleanups (Beckman Coulter) can be used to purify the sample by removing unused oligonucleotides and changing buffer composition between steps.

Figure 1. The xGen HS EGFR Pathway Amplicon Panels are used to prepare indexed Illumina®-compatible libraries from cfDNA or FFPE DNA. The workflow for preparing the final targeting sequencing libraries includes PCR I, PCR II, and PCR III. The first amplification uses the panel of primers to amplify the targeted genes from the EGFR pathway. The primers also incorporate molecular identifiers (MIDs). A second amplification step increases the total number of fragments for indexing, while the third PCR completes the indexing.

Product data

To evaluate the xGen HS EGFR Pathway Amplicon Panel, two DNA standards were used from Seracare, one with a 0.5% allele frequency for the 7 variants, and one with a 0.25% allele frequency. Duplicate SeraSeq® ctDNA Reference Material AF (LGC SeraCare) samples at 10 ng were generated with the 0.5% standard and duplicate SeraSeq® ctDNA Reference Material AF (LGC SeraCare) samples at 20 ng were generated with the 0.25% standard, a total of 4 libraries shown. As seen in Table 1, the observed allele frequency (AF) aligned with the expected allele frequency for the mutations in the reference cfDNA that were targeted by the xGen HS EGFR Pathway Amplicon Panel. Samples were sequenced on a MiniSeqTM (Illumina).

Table 1: Observed vs. expected allele frequency in NGS libraries prepared using the xGen HS EGFR Pathway Amplicon Panel

        10 ng SeraSeq cfDNA with AF of 0.5% 20 ng SeraSeq cfDNA with AF of 0.25%
Gene Mutation Chr Position Expected AF Observed AF (Rep 1) Observed AF (Rep 2) Expected AF Observed AF (Rep 1) Observed AF (Rep 2)
BRAF p.V600E 7 140453136 0.5% 0.19% 0.45% 0.25% 0.18% 0.18%
EGFR p.T790M 7 55249071 0.5% 0.43% 0.57% 0.25% 0.33% 0.36%
EGFR p.L858R 7 55259515 0.5% 0.86% 0.43% 0.25% 0.26% 0.25%
KRAS p.G12D 12 25398284 0.5% 0.59% 0.47% 0.25% 0.20% 0.48%
NRAS p.Q61R 1 115256529 0.5% 0.91% 0.56% 0.25% 0.30% ND
EGFR p.E746-A750 (delELREA) 7 55242265 0.5% 0.24% 0.61% 0.25% 0.11% 0.07%
EGFR p.D770-N771 (ins) 7 55249012 0.5% 0.43% 0.57% 0.25% 0.32% 0.36%

*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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