EGFR MET amplicons

Overview

The Epidermal Growth Factor Receptor (EGFR) gene plays a critical role in the control of cell proliferation, differentiation, and survival. Abnormalities in EGFR-related pathways have been linked to a range of cancers, including lung cancer. The MET (Mesenchymal Epithelial Transition Factor) gene is involved in abnormal activation of one of the EGFR-related pathways, triggering tumor growth, blood vessel proliferation, and metastasis.

This page describes a method, whereby the EGRF and MET genes are selectively amplified using the Fluidigm EGFR Gene Panel kit and Access Array System and the amplicons are subsequently converted into SMRTbells&tm; and sequenced on the PacBio® RS.

This study demonstrates the compatibility of the Fluidigm and PacBio systems and the ability of the PacBio RS to successfully detect known variants in well-characterized samples.

Methods

The Fluidigm® Access Array™ System was used to amplify 48 amplicons in each of two Coriell genomics DNA samples – NA17316 and NA17317. The 48 amplicons covered exons 2-28 in EGFR and exons 1-20 in MET. They ranged from 110bp to 317bp.

The amplicons were generated using the Fluidigm EGFR Gene Panel and Fluidigm’s Access Array™ IFC 4-Primer Amplicon Tagging Workflow. A second amplification involving 16 cycles was performed to generate the material needed for SMRTbell generation and libraries were constructed using the standard A-tailed library prep protocol.

Each sample was sequenced on a single SMRT® Cell on the PacBio RS instrument using the Standard Sequencing protocol with version 1 chemistry. Alignment against the EGFR-MET exons was performed using PacBio’s De Novo Circular Consensus Sequencing protocol. Data from the 4 SMRT Cells had the following characteristics:

• of mapped reads: 17,654 per SMRT Cell

• Mean mapped readlength: 2,245 bases
• 95th percentile readlength: 4,273 bases
• Mean depth of coverage: 156
• Consensus accuracy: 100%, except for true positive SNPs
• Mean single molecule raw accuracy: 98.2% (mode 99.15%)

Using Sanger sequencing, we validated:

• 4 homozygous SNPs
  • 1 in NA17316
  • 3 in NA17317
• 10 heterozygous SNPs
  • 2 in NA17316
  • 8 in NA17317

Technical replicates were in agreement for all SNP calls.

Conclusion

The Fluidigm Access Array system for the generation of amplicons is compatible with sequencing on the PacBio RS. De Novo Circular Consensus Sequencing successfully detected known variants in two Coriell samples.