Results and Applications

Improved sensitivity of NGS

OnTarget™ improves the sensitivity of next-generation sequencing (NGS) platforms to below 0.01% mutant content. In the following experiment, titrated PIK3CA E454K mutant and wild-type samples were sequenced using MiSeq® alone and a combined OnTarget™ + MiSeq® assay.  For the 0.01% mutant sample, MiSeq® alone cannot distinguish with any statistical confidence between the 0.01% PIK3CA E454K mutant and the wild-type sample; while the combined OnTarget™ + MiSeq® assay can clearly identify the 0.01% mutant signal as many standard deviations greater than the very low background of the wild-type sample. This combined assay enables highly sensitive mutation detection below 0.01% mutant content with >99.9% specificity.

Quantification of rare mutations in plasma

In the following experiment, OnTarget™ enrichment was combined with SNaPshot®, a single base extension mutation detection assay. The combined workflow was demonstrated to robustly detect BRAF V600E mutant DNA at abundances at or below 0.03%, over 2 orders of magnitude lower than the limit of detection of SNaPshot® alone (~5%), which on its own shows no detectable mutant signal for any of these samples.

The combined OnTarget™ + SNaPshot® assay is sensitive to as little as a single mutant input copy. The titrated 0.03% mutation content samples contain an average of 6 mutant copies, and 5.5 ± 2.5 copies were detected as expected from statistical fluctuation. This is well above the counts of 0.05 ± 0.06 copies detected in wild-type only and 0.1 ± 0.1 copies in no-template control (NTC) samples, which are thus determined to contain less than 1 copy of mutant to greater than 99.99% confidence.

Experimental details: DNA was extracted from plasma of a cancer patient carrying the BRAF V600E mutation and plasma with wild-type BRAF. For accurate titration, DNA was extracted and its concentration and mutation abundance (10% for the mutant DNA) were measured prior to mixing. Following titration, replicates containing 60 ng total DNA (18,000 copies) were used for OnTarget™ + SNaPshot® and SNaPshot® only workflows. Multiple replicates were used for each mutation abundance and NTC.

Highly sensitive detection from FFPE

In this experiment, OnTarget™ was again combined with the SNaPshot® mutation detection assay. The combined OnTarget™ + SNaPshot® assay demonstrated robust detection of BRAF V600E mutant DNA at abundances at or below 0.1%, nearly 2 orders of magnitude lower than the limit of detection of SNaPshot® alone (~5%), which on its own shows no detectable mutant signal for any of these samples.

OnTarget™ + SNaPshot® detects 23 ± 7 copies in the 0.1% sample as expected from statistical fluctuation. This is well above the counts of 0.2 ± 0.02 copies detected in no-template control (NTC) and 0.6 ± 0.2 copies in wild-type only samples, which are thus determined to contain less than 1 copy of mutant to greater than 99% confidence.

Experimental details: DNA was extracted from tumor FFPE from a patient carrying the BRAF V600E mutation and FFPE with wild-type BRAF. For accurate titration, DNA was extracted and its concentration and mutation abundance (30% for the mutant DNA) were measured prior to mixing. Following titration, replicates containing 100 ng total DNA (18,000 copies assayed by qPCR) were used for OnTarget™ + SNaPshot® and SNaPshot® only workflows. Multiple replicates were used for each mutation abundance and NTC.