HEAT-Seq Ultra Oncology HotSpot Panel

The HEAT-Seq® Ultra Oncology HotSpot Panel is based on amplification-based enrichment employing an advanced version of Molecular Inversion Probe (MIP) technology and combines a fast and easy workflow with powerful SNV detection. The panel contains 635 probes, and has a capture target of approximately 30.5 Kb across previously described mutation hotspots in 53 commonly targeted oncogenes and is ideal for detecting and characterizing low-frequency mutations. The panel helps with the detection of variants at 1% minor allele frequency (MAF) and below.

Benefits of Heat-Seq Oncology HotSpot Panel

  • Smaller overall design size with simplified analysis and high throughput
  • Detection and removal of PCR duplicates using molecular barcoding and the ability to confidently discern  a biological replicate and a process artifact
  • Maximized variant detection over a wide range of allelic frequencies using molecular barcoding with UIDs without the need for deep sequencing
  • Simple workflow with sequencing-ready DNA in a single tube with just two hours of hands-on time
  • Minimized sequencing errors due to the stringent removal of false positives
  • Utilize HSQutils software, which is a separately sourced sequencing read mapper and variant caller for use in the analysis workflow

Heat-Seq Workflow

The final product of the workflow is a sequencing-ready amplicon that contains the target DNA sequence, a molecular barcode, and sample indices.

The entire workflow is completed in 8 hours with < 2 hours of hands-on time using a single tube. The protocol is easy to adopt and is also automation-friendly.

 

Product Highlights

Target the mutation hotspots in oncogenes with one panel 

HEAT-Seq Ultra Oncology Panel genes

UABL1

EZH2

KDR

PDGFRA

AKT1

FBXW7

KIT

PIK3CA

ALK

FGFR1

KRAS

PTEN

APC

FGFR2

MAP2K1

PTPN11

ATM

FGFR3

MAP2k4

RB1

BRAF

FLT3

MET

RET

BRCA1

GNA11

MLH1

RUNX1

BRCA2

GNAQ

MPL

SMAD4

CDH1

GNAS

MSH2

SMARCB1

CDKN2A

HNF1A

MSH6

SMO

CSF1R

HRAS

NF1

SRC

CTNNB1

IDH1

NF2

STK11

EGFR

IDH2

NOTCH1

TERT

ERBB2

JAK2

NPM1

TP53

ERBB4

JAK3

NRAS

VHL

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Uniform and complete coverage of the capture target

HEAT-Seq Ultra Oncology HotSpot Panel performance

The hotspot panel was tested using 100 ng of FFPE DNA. 24 samples were multiplexed on a single MiSeq lane using v3 chemistry. Data from three replicate experiments is shown. High on-target coverage, uniformity, and coverage rates were observed for each replicate.

Accurate and sensitive detection of allele frequency

The HEAT-Seq Ultra Oncology HotSpot Panel was tested using an FFPE control sample with known mutations at frequencies between 0.9% and 25%. Observed allele frequencies strongly matched the known frequencies, showing the sensitivity and accuracy of this panel..

High coverage depth over regions of interest

Coverage depth over regions of interest.

The HEAT-Seq Ultra Oncology HotSpot panel was tested using the Formalin Compromised Quantitative Multiplex Reference Standard (HD-C749, Horizon Diagnostics). Using all available reads (3-5 M per sample), the panel provided coverage of between 250x and 1250x of known mutations.

Sensitive detection of false positives

False-positive rate in detection of variants using various methods.

The performance of the HEAT-Seq Ultra Oncology HotSpot Panel is compared to similar kits to assess allele frequency detection accuracy and data quality. The HEAT-Seq panel presents a similar or improved variant detection ability, but also presents an extremely low false-positive rate. The proprietary process of removing duplicates using molecular barcoding is the core of the HEAT-Seq system that enables not only accurate variant calls but also the confident removal of process induced errors that appear as false positives. 

  HEAT-Seq Ultra Oncology HotSpot Panel
Illumina TruSeq Amplicon Cancer Panel
Thermo Fisher Ion AmpliSeq Cancer Panel v2
Average False-Positive Rate 0.014% 0.213% 3.071%
Range 0.0% - 0.2% 0.0% - 0.4% 0.0 - 11.1%
Missed Calls 0 2 0

Design Files

The design and annotation files provide information about genomic regions covered by the capture probes and the genes included in these regions.

These files were designed for use with the following Roche products:

  • HEAT-Seq Ultra Oncology Panel, 24 Reactions (Catalog No. 07757069001)
  • HEAT-Seq Ultra Oncology Panel, 96 Reactions (Catalog No. 07768613001)

Download Design File

Demo Data

Demo data is available for evaluation. Please contact Roche representative for your country.