Ion Proton amplicons for clinical molecular diagnostics

A recent paper from the MD Anderson's Department of Hematopathology reports on their use of the AmpliSeq for a 409 gene panel on Ion Proton: Clinical massively parallel next-generation sequencing analysis of 409 cancer-related genes for mutations and copy number variations in solid tumours. I'm a big fan of amplicomes, another 'ome I know, and in this context used to mean all the amplicons in your panel. PCR is a great way to enrich for specific regions of the genome and we all understand the basics. For me amplicomes on NGS is perhaps the easiest NGS for the NHS. It simply replaces M13-PCR for a single amplicon with a multiplex PCR, and Sanger sequencing with NGS. If a doctor understood a Sanger test then this is perhaps an easier leap to make than trying to understand/explain what an exome is!

Back to the paper:  The MD Anderson group used their gene panel on FFPE material and reported on the "sensitivity, specificity, reproducibility, and applicability of using the Ion Proton 409-gene panel to routinely screen for SNVs, insertions/deletions, and CNVs". To do this they used 55 tumours (20 with paired normals) and four cell lines.

Fig 6A: ERBB2 amplifications as seen in 4 breast cancer samples.

The Ion Torrent Ampliseq Comprehensive Cancer Panel uses 4000 primer pairs across four primer pools each requiring just 15ng on input DNA (60ng total), and up to 96 samples can be indexed using Ion Xpress Barcodes. Alignment and analysis of data was performed using the Torrent Suite software. And the groups OncoSeek was used to generate a clinical report.

Concordance between platforms was high when the group compared results to an earlier PGM panel of 46 genes, and detected pretty much everything they expected. The Proton did call some InDels that the PGM missed, which the group put down to improvements in calling software.

The group reported high sensitivity for SNVs up to 5% allelic fraction, and high reproducibility. This, coupled to the minimal FFPE input, and fast turnaround (5 days) makes the platform combination one that could be used in a clinical molecular diagnostics laboratory. Additionally they reported that up to 10 samples could be multiplexed per run and that this fitted in particularly well with their workflows.