Performance characteristics of the AmpliSeq Cancer Hotspot panel v2 in combination with the Ion Torrent Next Generation Sequencing Personal Genome Machine

Abstract

Next-Generation Sequencing is a rapidly advancing technology that has research and clinical applications. For many cancers, it is important to know the precise mutation(s) present, as specific mutations could indicate or contra-indicate certain treatments as well as be indicative of prognosis. Using the Ion Torrent Personal Genome Machine and the AmpliSeq Cancer Hotspot panel v2, we sequenced two pancreatic cancer cell lines, BxPC-3 and HPAF-II, alone or in mixtures, to determine the error rate, sensitivity, and reproducibility of this system. The system resulted in coverage averaging 2000× across the various amplicons and was able to reliably and reproducibly identify mutations present at a rate of 5%. Identification of mutations present at a lower rate was possible by altering the parameters by which calls were made, but with an increase in erroneous, low-level calls. The panel was able to identify known mutations in these cell lines that are present in the COSMIC database. In addition, other, novel mutations were also identified that may prove clinically useful. The system was assessed for systematic errors such as homopolymer effects, end of amplicon effects and patterns in NO CALL sequence. Overall, the system is adequate at identifying the known, targeted mutations in the panel.

Application of a multi‐gene next‐generation sequencing panel to a non‐invasive oesophageal cell‐sampling device to diagnose dysplastic Barrett's oesophagus

“…These factors led us to sequence at high depth (1000×) and great care was taken in defining the cut‐off for calling a mutation. Previous studies have found that mutations can accurately be called if they have an AF of 5% or greater . The lowest AF mutation which we confirmed was present at 1.7%.…”

“…TP53 coverage was used for quality control and samples with coverage <100× for each exon were considered to have failed. This reflected the coverage of all mutations and previous studies have considered a coverage of >100× to be adequate .…”

Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer

“…Anyway, due to the Ion Torrent chemistry, the homopolymer sequencing errors are known to be an issue, but the examination of our experimental results did not reveal any homopolymer-based errors or any errors caused by the position of the mutation in the amplicon. This may be due to the design of the primers used in the Cancer Hot Spot panel v2 or the location of the mutations [ 31 – 35 ].…”

Comparative Study of NGS Platform Ion Torrent Personal Genome Machine and Therascreen Rotor-Gene Q for the Detection of Somatic Variants in Cancer

“…For the identification of mutation profile in cancer patients, it is necessary to use alternative methods of analysis with different percentages of accuracy and sensitivity to detect a mutation-driven drug resistance. Ion Torrent Personal Genome Machine has a sensitivity of results of 95% [28], while the qualitative real time PCR Rotor gene Q has a sensitivity of 99%. In our cohort of patients, the use of the two methods allowed us to identify mutations that otherwise we would have not found, with negative consequences on the clinical choices for the patients.…”