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Accelerating ScienceBehind the Bench / Cancer Research / How do you check for quality for a next-generation sequencing cancer research panel?

How do you check for quality for a next-generation sequencing cancer research panel?

By 10.16.2014

When researching retrospective cancer samples, a prime problem is the inherent heterogeneity of the starting material. Many experiments must be run in order to measure the performance of the system; in particular its sensitivity to pick up known mutations, whether single-nucleotide variants (SNVs), multiple nucleotide variants (MNVs), or insertions / deletions (what are called ‘indels’ collectively). Thus a researcher has to maintain known cell-line DNAs, the specific mutations in those cell lines for the genes or regions of interest, and then run a titration series with known background DNAs and analyze the data.

A titration series is run in order to determine what kind of sensitivity a given mutation in a sample is detected from a normal sample background; i.e. to test a 5% minor allele frequency (MAF) at a greater than 10x coverage for that minor allele mutation, the cell-line DNA would be mixed with known background DNA at a 1:20 ratio for a 5% frequency, and other percentages as desired.

Given that the Ion AmpliSeq™ Cancer Hotspot Panel v2 covers some 2800 COSMIC mutations from over 50 genes, where does one begin?

genes.pngThermo Fisher Scientific has a line of quality control reagents under the AcroMetrix® brand to assist in the implementation and demonstration of assay controls for clinical research laboratories and manufacturers. These controls have previously been Sanger sequencing-based and TaqMan Real-Time PCR-based, and recently the AcroMetrix® Oncology Hotspot Control which is NGS-based. And recently Thermo Fisher made available the AcroMetrix® Oncology Hotspot Control, designed for high-multiplex NGS detection of somatic mutations in your research. This product was designed for high-multiplex NGS detection of somatic mutations in research samples.

These controls are synthetic stretches of DNA sequences from particular cancer hotspot genes that are mixed in a precise ratio to a larger background of a nominal genomic DNA sample. There are 53 genes included in this control that cover some 555 COSMIC mutations.

 

Some of the synthetic control DNA is present in a lower amount (a target frequency of 5 to 15%), while others are higher in amount (15 to 35%), and other detected mutations are from the background ‘normal’ DNA. (The background DNA used is Coriell Accession GM24385, which harbors some mutations picked up by different research panels.) It is worthwhile to note that each laboratory is responsible for establishing its chart 1.pngown expected results.

As a genomic DNA control, this can serve many purposes: from observing variation in procedures, measuring run-to-run consistency, or help troubleshoot parameters in analysis. Since this product is genomic DNA at a known concentration, it can be used for any platform or enrichment strategy, in front of any next-generation sequencing platform. This product is manufactured like other AcroMetrix® products, in accordance to ISO 17511, traceable to a quality standard. The synthetic variants range in size from 1 nucleotide to 40 nucleotides, and all have been confirmed by Sanger sequencing.

 

For further information, please visit the product page for the AcroMetrix® Oncology Hotspot Control. If you are interested, please learn more about all of our quality controls and standards for clinical research.

TaqMan® is a registered trademark of Roche Molecular Systems, Inc. used under permission and license.

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