Optimizing NGS for NTRK Gene Fusions

NGS Overview

Identifying NTRK gene fusions with NGS

Many commercially available NGS kits can detect NTRK gene fusions. NGS enables sequencing of multiple nucleic acid targets in a rapid and massively parallel manner.1 This method is ideal because it addresses the unique multiple partner and variable structural properties of NTRK gene fusions.2

NGS can address the structural characteristics of NTRK gene fusions; thus, many commercially available kits for NTRK gene fusion detection are NGS-based2,3

Review NTRK gene fusion structural characteristics

DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; RNA, ribonucleic acid.

References:

Gagan J, Van Allen EM. Next-generation sequencing to guide cancer therapy.Genome Med.2015;7(1):80. 2. Murphy DA, Ely HA, Shoemaker R, et al. Detecting gene rearrangements in patient populations through a 2-step diagnostic test comprised of rapid IHC enrichment followed by sensitive next-generation sequencing. Appl Immunohistochem Mol Morphol. 2017;25(7):513-523. 3. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. Return to content

RNA- vs DNA-based NGS

RNA- vs DNA-based NGS

RNA-based NGS methods are preferred for the detection of NTRK gene fusions1-3

RNA-based NGS sequences transcribed and spliced mature mRNAs (with introns already removed)3
Thus, RNA-based NGS avoids the difficulties associated with sequencing through challenging features of NTRK gene fusions and is able to identify in-frame fusion products2,3,5

General NGS

High sensitivity and specificity potential1,2
Multiplexing: simultaneously queries multiple potentially actionable targets4 (eg, ALK, ROS1, RET)
Detects both known and novel fusions, regardless of breakpoint or fusion partner (depending on the library prep method)2,5

RNA-Based NGS

Able to distinguish in-frame, transcribed gene fusions vs out-of-frame fusions2,5
Avoids difficulties of sequencing large intronic regions in the NTRK genes3

PLANNING FOR YOUR PRACTICE

Appropriately designed RNA-based NGS provides optimal method to detect NTRK gene fusions since no prior knowledge of fusion breakpoint and/or fusion partner is required and addresses the unique structural challenges present in NTRK gene fusions3

Watch Dr Andrew Turk discuss the considerations for using RNA- vs DNA-based NGS testing to detect NTRK gene fusions

ALK, anaplastic lymphoma kinase; DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; mRNA, messenger ribonucleic acid; RET, Ret proto-oncogene; RNA, ribonucleic acid; ROS1, ROS proto-oncogene 1.

References:

Murphy DA, Ely HA, Shoemaker R, et al. Detecting gene rearrangements in patient populations through a 2-step diagnostic test comprised of rapid IHC enrichment followed by sensitive next-generation sequencing.Appl Immunohistochem Mol Morphol. 2017(7);25:513-523. Return to content
Serrati S, De Summa S, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. Onco Targets Ther. 2016;9:7355-7365. Return to content
Abel H, Pfeifer J, Duncavage E. Translocation detection using next-generation sequencing. In: Kulkarni S, Pfeifer J, eds. Clinical Genomics. New York, NY: Elsevier/Academic Press; 2015:151-164. Return to content
Farago AF, Azzoli CG. Beyond ALK and ROS1: RET, NTRK, EGFR and BRAF gene rearrangements in non-small cell lung cancer. Transl Lung Cancer Res. 2017;6(5):550-559. Return to content
Schram AM, Chang MT, Jonsson P, Drilon A. Nat Rev Clin Oncol. 2017;14(12):735-748. Return to content

NGS Methodologies

NGS Methodologies

There are 5 common NGS approaches for sequence data collection1-4

Choice of sequencing methodology is determined by the genomic regions and variant types of interest. Subsequently, this informs selection of NGS platform, NGS panel, bioinformatic analysis pipeline, and lab resources to support in-house verification and validation of NGS testing.3

Whole-genome sequencing

Sequences the majority of the genome, including coding (exons) and noncoding (introns) genomic regions⁵
Example: Illumina Nextera DNA Flex Library Prep Kit for whole-genome sequencing6,7

Whole-exome sequencing

Targets the coding (exon) region of an individual’s genome5
Example: Thermofisher Ion AmpliSeq™ Exome RDY Kit8

Whole-transcriptome sequencing

Targets mRNA transcripts. Enables determination of expression, quantity, and presence of alterations in the nucleic acid sequence of an individual’s genome⁵
Example : Illumina RNAseq Total Transcriptome Kit9,10

Targeted DNA/RNA sequencing

Targets selected genes of interest, focusing on those that are clinically meaningful and actionable (eg, EGFR, BRAF, KRAS, etc) for genomic alterations that include SNVs, indels, CNVs, and fusions⁵
Example : ThermoFisher Oncomine Comprehensive Assay11,12

Targeted hotspot sequencing

Targets frequently mutated “hotspot” regions of genes to detect well-known SNVs, indels, and gene fusions (if breakpoints and partners are known)¹³
Example : ThermoFisher Ion AmpliSeq™ Cancer Hotspot Panel v2 (does not include NTRK1, NTRK2, NTRK3)14-16

PLANNING FOR YOUR PRACTICE

Targeted RNA-based sequencing, or whole-exome/transcriptome sequencing are preferred, as they may be more suited to address some of the challenges in detecting NTRK gene fusions5

Watch Dr Alexander Lazar delineate the key distinctions among 5 common NGS approaches for sequence data collection

BRAF, v-raf murine sarcoma viral oncogene homolog B1; CNVs, copy number variations; DNA, deoxyribonucleic acid; EGFR, endothelial growth factor receptor; KRAS; Kirsten rat sarcoma viral oncogene homolog; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; mRNA, messenger ribonucleic acid; RNA, ribonucleic acid; SNV, single nucleotide variant.

References:

Wang Q, Xia J, Jia P, Pao W, Zhao Z. Application of next generation sequencing to human gene fusion detection: computational tools, features and perspectives. Brief Bioinform. 2013;14(4):506-519. Return to content
Dong L, Wang W, Li A, et al. Curr Genomics. 2015;16(4):253-263. Return to content
Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. Return to content
Basho RK, Eterovic AK, Meric-Bernstam F. Clinical applications and limitations of next-generation sequencing. Am J Hematol Oncol. 2015;11(3):17-22. Return to content
Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. Return to content
Illumina. Nextera DNA Flex Library Prep Kit. https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-dna-flex.html. Accessed March 27, 2019. Return to content
Illumina. TruSight HLA v2 Sequencing Panel. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/trusight-hla-v2-data-sheet-070-2016-002.pdf. Accessed March 27, 2019. Return to content
Thermo Fisher Scientific. Ion AmpliSeq™ Exome RDY Kit 4X2. https://www.thermofisher.com/order/catalog/product/A38264. Accessed March 27, 2019. Return to content
Illumina. Study gene expression using RNA sequencing. https://www.illumina.com/techniques/sequencing/rna-sequencing.html. Accessed March 27, 2019. Return to content
Illumina. A comprehensive picture of the transcriptome. https://www.illumina.com/techniques/sequencing/rna-sequencing/total-rna-seq.html. Accessed March 27, 2019. Return to content
Ion Torrent. Oncomine comprehensive assay v3 flyer. https://tools.thermofisher.com/content/sfs/brochures/oncomine-comprehensive-assay-v3-flyer.pdf. Accessed March 27, 2019. Return to content
Thermo Fisher Scientific. Ion Torrent. Oncomine comprehensive assays. https://www.thermofisher.com/us/en/home/clinical/preclinical-companion-diagnostic-development/oncomine-oncology/oncomine-cancer-research-panel-workflow.html. Accessed March 27, 2019. Return to content
Oliveira DM, Mirante T, Mignogna C, et al. Simultaneous identification of clinically relevant single nucleotide variants, copy number alterations and gene fusions in solid tumors by targeted next-generation sequencing. Oncotarget. 2018;9(32):22749-22768. Return to content
Thermo Fisher Scientific. Ion AmpliSeq cancer hotspot panel v2. https://www.thermofisher.com/order/catalog/product/4475346. Accessed March 27, 2019. Return to content
Thermo Fisher Scientific. Ion AmpliSeq cancer hotspot panel v2. https://assets.thermofisher.com/TFS-Assets/LSG/brochures/Ion-AmpliSeq-Cancer-Hotspot-Panel-Flyer.pdf. Accessed March 27, 2019. Return to content
Thermo Fisher Scientific. Ion Torrent. Ion AmpliSeq panels for focused next-generation sequencing flyer. https://assets.thermofisher.com/TFS-Assets/CSD/Flyers/flyer-ampliseq-ampliseq-hd.pdf. Accessed March 27, 2019. Return to content

NGS Workflow Overview

NGS Workflow Overview

Optimizing NGS workflow at every step is required to enable robust testing and reporting1

Preanalytic phase

NUCLEIC ACID EXTRACTION: SAMPLE

Using tissue from biopsy or blood sample, obtain purified DNA or RNA to be enriched and sequenced1

Review key considerations for sample handling

Analytic phase

LIBRARY PREP: PANEL

Select genes/regions to sequence (from a few to hundreds)1
Perform target enrichment (via hybrid capture or PCR) to select and amplify regions of interest1
Perform clonal amplification (if required) to ensure sufficient material for successful sequencing1

SEQUENCING: DNA SEQUENCER

Determines the sequence of nucleotides in the nucleic acid sample tested1

Learn more about NGS library preparation and target enrichment

Postanalytic phase: Bioinformatics

ANALYSIS

Separates unique patients by barcode2
Aligns sequence reads to a reference sequence1
Filters noise, repeated sequences1
Identifies variations1
Determines what is potentially clinically meaningful vs usual human variation1

ANNOTATION AND REPORT GENERATION

PLANNING FOR YOUR PRACTICE

Understanding the stepwise approach outlined in this section can help streamline the NGS testing process to optimize conditions for NTRK gene fusion detection1

Explore an example journey for RNA-based NGS testing at the Providence St. Joseph Health Molecular Genomics Laboratory

DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; PCR, polymerase chain reaction; RNA, ribonucleic acid.

References:

Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. Return to content
Aziz N, Zhao Q, Bry L, et al. College of American Pathologists’ laboratory standards for next-generation sequencing clinical tests. Arch Pathol Lab Med. 2015;139(4):481-493. Return to content

Preanalytic Phase

Preanalytic Phase

Detection of genomic alterations using NGS is significantly affected by quality of starting sample material1

It is critical for the anatomical pathologist to assess tumor samples under a microscope before the sample can be accepted for NGS testing. During this time, it’s also important that the sample is enriched by macro- or microdissection techniques, even when the relative proportion of tumor nuclei is prevalent at high frequency.1

Key considerations for sample handling

Heterogeneity
- Ensuring sufficient tumor cell content compared to normal cells1
- Differing subclone populations may result in different mutation combinations
Size of sample1
- Biopsying technique may limit amount of DNA/RNA available1
Preprocessing
- Formalin fixation can create random artificial mutations that may result in false positives when using a highly sensitive technique like NGS1

PLANNING FOR YOUR PRACTICE

Preanalytical review of the sample and tumor enrichment by microdissection helps ensure sufficient tumor for analysis and increases sensitivity for the gene alteration1

Explore sample considerations across methodologies

DNA, deoxyribonucleic acid; NGS, next-generation sequencing; RNA, ribonucleic acid.

Reference:

Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing–based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists.J Mol Diagn. 2017;19(3):341-365. Return to content

Analytic Phase

Analytic Phase

Library prep is a critical step in the NGS process1

Library prep generates a pool of sequencing targets representing areas of interest ready for sequencing on an appropriate NGS platform. Target enrichment is a key component of this step. During target enrichment, DNA or RNA isolated from a sample is enriched for regions of interest and formatted for sequencing on the chosen platform.1

Choice of target enrichment method during library prep is a key consideration when detecting NTRK gene fusions2,3

Two potential methods for target enrichment are amplification and hybridization. Both methods have unique attributes that can influence their ability to optimally detect various genomic alterations.1

Amplification vs Hybridization-based Capture
Amplification		Hybridization-Based Capture
Variants Detected	Best suited for detecting known variants of interest (SNVs or indels)4 Can include known, specific gene fusions (eg, EML4-ALK) and CNVs1	Potential for broad detection of all actionable mutations (CNVs, SNVs, indels, and fusions) depending on assay design, potentially including novel gene fusion partners¹
Benefits	Commonly used for small panels; ideal for SNVs, indels, and known fusions¹ Requires low input of nucleic acid1	Can detect larger number of genes and genomic regions1 Sensitivity for detecting fusions with novel fusion partners¹
Drawbacks	Requires complex amplicon design to sufficiently cover all genomic alterations including gene fusions¹	Requires more input of nucleic acid than amplification-based methods¹

Amplification vs Hybridization-based Capture
Amplification
Variants Detected	Best suited for detecting known variants of interest (SNVs or indels)4 Can include known, specific gene fusions (eg, EML4-ALK) and CNVs1
Benefits	Commonly used for small panels; ideal for SNVs, indels, and known fusions¹ Requires low input of nucleic acid1
Drawbacks	Requires more input of nucleic acid than amplification-based methods¹

Hybridization-Based Capture
Variants Detected	Potential for broad detection of all actionable mutations (CNVs, SNVs, indels, and fusions) depending on assay design, potentially including novel gene fusion partners¹
Benefits	Can detect larger number of genes and genomic regions1 Sensitivity for detecting fusions with novel fusion partners¹
Drawbacks	Requires more input of nucleic acid than amplification-based methods¹

PLANNING FOR YOUR PRACTICE

Hybridization-based capture approach may be better than amplicon-based approach as this method can detect a broad range of NTRK gene fusions (known and novel)1-3,5

Clonal amplification generates required copies of templates of interest for accurate sequencing1,6

Depending on NGS platform, clonal amplification may be required to ensure sufficient material for successful sequencing.1

Clonal amplification approach with Thermo Fisher Ion Personal Genome Machine, Thermo Fisher Proton, or Qiagen GeneReader6-8

Clonal amplification approach of emulsion, on-bead amplification, and final product graphic

Clonal amplification approach with Illumina MiSeq or Illumina NextSeq6,9

Clonal amplification approach of template binding step, bridge PCR amplification, and cluster generation graphic

No clonal amplification needed with PacBio RS 1/II, Oxford nanopore, or GridlON6,10,11

Find commercially available NGS kits

Current NGS technologies employ different sequencing methods6

SEQUENCING BY SYNTHESIS USING SERIAL DNTP FLOW (THERMOFISHER SCIENTIFIC): SEMICONDUCTOR SEQUENCING6

Template-enriched beads are carefully arrayed into a microtitre plate chip6
Each of the 4 DNA nucleotide species is added iteratively to ensure only 1 dNTP is responsible for the signal6
As each base is incorporated, a small unit change in pH is detected by an integrated CMOS–ISFET sensor device6
The pH change detected by the sensor is proportional to the number of nucleotides detected6
After signal detection and recording, unincorporated bases are washed away and the next one is added to repeat the process6

SEQUENCING BY SYNTHESIS USING REVERSIBLE TERMINATORS (ILLUMINA AND QIAGEN)6

Primers, DNA polymerase, and modified nucleotides added to template-enriched clusters (Illumina) or beads (Qiagen) on flow cell6
Nucleotide addition
- All 4 uniquely fluorophore-labeled, terminally blocked nucleotides added and hybridize to complementary base. Each cluster incorporates a different base (Illumina)6 or
- All 4 uniquely fluorophore-labeled, terminally blocked and unlabeled, blocked nucleotides added and hybridize to complementary base. Each bead incorporates a different base (Qiagen)6
Imaging
- Slides are imaged with 2 or 4 laser channels. Each cluster emits color corresponding to base incorporated during cycle (Illumina)6 or
- Slides are imaged with 4 laser channels. Each bead emits color corresponding to base incorporated during cycle (Qiagen)6
Cleavage
- Fluorophores cleaved and washed away. A new cycle begins with the addition of new nucleotides6

NGS platform considerations12-18

MANUFACTURER	METHOD	INSTRUMENT	RUN TIME	READ LENGTH	OUTPUT (Gb)
Illumina	Bridge PCR Chemistry: hybridization Sequence by synthesis Paired end sequencing	MiSeq v2 MiSeq v3 NextSeq HiSeq	~39 hours ~56 hours ≤35 hours 6 days	2x250 2x300 2x150 2x125	7.5-8.5 13.2-15 ≥90 450-500
ThermoFisher	Emulsion PCR Semiconductor sequencing Single end sequencing	Ion PGM (CE/IVD) Ion Proton Ion S5	5 hours 2.5 hours 12 hours	400 200 400	0.6-1 15 1.2-2
Qiagen	Emulsion PCR Sequencing by synthesis Single end sequencing	GeneReader	30 hours	134	1

MANUFACTURER	METHOD
Illumina	Bridge PCR Chemistry: hybridization Sequence by synthesis Paired end sequencing
ThermoFisher	Emulsion PCR Semiconductor sequencing Single end sequencing
Qiagen	Emulsion PCR Sequencing by synthesis Single end sequencing

MANUFACTURER	INSTRUMENT	RUN TIME
Illumina	MiSeq v2 MiSeq v3 NextSeq HiSeq	~39 hours ~56 hours ≤35 hours 6 days
ThermoFisher	Ion PGM (CE/IVD) Ion Proton Ion S5	5 hours 2.5 hours 12 hours
Qiagen	GeneReader	30 hours

MANUFACTURER	READ LENGTH	OUTPUT (Gb)
Illumina	2x250 2x300 2x150 2x125	7.5-8.5 13.2-15 ≥90 450-500
ThermoFisher	400 200 400	0.6-1 15 1.2-2
Qiagen	134	1

PLANNING FOR YOUR PRACTICE

To ensure a testing method is most appropriate for your existing laboratory workflow, considerations for choosing an NGS platform and a commercial kit should be made together

Review key attributes of commercially available NGS kits

Watch Dr Michelle Shiller compare target enrichment techniques for NTRK gene fusion detection

CNV, copy number variations; DNA, deoxyribonucleic acid; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; PCR, polymerase chain reaction; RNA, ribonucleic acid; SNV, single nucleotide variant; ssDNA, single-strand DNA.

References:

Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing–based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. Return to content
Serrati S, De Summa S, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. Onco Targets Ther. 2016;9:7355-7365. Return to content
Kummar S, Lassen UN. Target Oncol.2018;13(5):545-556. Return to content
Vendrell JA, Taviaux S, Béganton B, et al. Detection of known and novel ALK fusion transcripts in lung cancer patients using next-generation sequencing approaches. Sci Rep. 2017;7(1):1-11. Return to content
Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. Return to content
Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genetics. 2016;17:333-351. Return to content
Platform manufacturer website: ThermoFisher Scientific. https://www.thermofisher.com/us/en/home/applications-techniques.html. Accessed February 27, 2019. Return to content
Platform manufacturer website: Qiagen. https://www.qiagen.com/us/products/ngs/ngs-life-sciences/dna-amplicon-sequencing/. Return to content
Platform manufacturer website: Illumina. https://www.illumina.com/techniques/sequencing/dna-sequencing.html. Accessed February 27, 2019. Return to content
Platform manufacturer website: PacBio. https://www.pacb.com/applications/rna-sequencing/. Accessed February 27, 2019. Return to content
Platform manufacturer website: Oxford Nanopore. https://nanoporetech.com/applications/basic-genome-research. Accessed February 27, 2019. Return to content
Platform manufacturer website: Illumina. Specifications for the MiSeq system. Cluster generation and sequencing. https://www.illumina.com/systems/sequencing-platforms/miseq/specifications.html. Accessed February 26, 2019. Return to content
Platform manufacturer website: Illumina. System specifications for NextSeq 550Dx. Performance parameters. https://www.illumina.com/systems/sequencing-platforms/nextseq-dx/specifications.html?langsel=/us/. Accessed February 26, 2019. Return to content
Platform manufacturer website: Illumine. Performance specifications for the HiSeq 2500 system. High output run mode. https://www.illumina.com/systems/sequencing-platforms/hiseq-2500/specifications.html. Accessed February 26, 2019. Return to content
Platform manufacturer website: ThermoFisher Scientific. Ion PGM™ Sequencer Specifications/Ion GM System specifications. https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-pgm-system-for-next-generation-sequencing/ion-pgm-system-specifications.html. Accessed February 26, 2019. Return to content
Platform manufacturer website: ThermoFisher Scientific. Ion Proton™ Sequencer Specification. Ion Proton System performance specifications with Ion PI Chip. https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-proton-system-for-next-generation-sequencing/ion-proton-system-specifications.html. Accessed February 26, 2019. Return to content
Platform manufacturer website: ThermoFisher Scientific. Ion GeneStudio S5 Spec. Flexibility in throughput and workflow for a broad range of NGS applications. https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-run-sequence/ion-s5-ngs-targeted-sequencing/ion-s5-specifications.html. Accessed February 26, 2019. Return to content
Platform manufacturer website: DeciBio. With the GeneReader launch Qiagen offers a true end-to-end clinical NGS solution.https://www.decibio.com/2015/11/06/with-the-genereader-launch-qiagen-offers-a-true-end-to-end-clinical-ngs-solution/. Accessed February 26, 2019. Return to content

Postanalytic Phase

Postanalytic Phase

Bioinformatics: Analyzing, interpreting, and reporting NGS data

Multiple bioinformatic tools available from sequencing providers and third-party vendors are evaluated, selected, and then integrated to create a validated bioinformatic process for NGS that allows for the discovery and identification of gene fusions.1

Bioinformatics workflow: Key processes for accurate gene fusion detection1

Sequencing data: Platform-specific DNA sequences determined and demultiplexed to produce pool of patient specific-sequence reads1
Alignment: Patient-specific sequence reads assembled or mapped by comparing to a control (reference) DNA genome sequence1
Variant calling: DNA base pairs in the reads or segments of the reads different from the reference are noted as variants1
Variant annotation: Variants are compared against public or proprietary databases to determine biological relevance1
Variant interpretation: Relevant variants compared against public or proprietary databases to determine and add clinical context to the annotation1
Sequencing report: Provides key findings and actionable steps to end-user (ie, triage patients to a clinical trial or prescribe a targeted therapy)1

PLANNING FOR YOUR PRACTICE

Choosing the appropriate bioinformatics tools directly impacts the diagnostic utility of sequencing by NGS. For this reason, sequencing data should be analyzed with bioinformatics processes that are optimized for the identification of NTRK gene fusions2

Experimental and bioinformatic considerations for carrying out RNA-based NGS

Number of reads

Total number of RNA transcript sequence reads should be optimized for system under study to support gene fusion detection³

Length of reads

Paired-end sequencing and longer sequence reads provide data that accumulate overlapping coverage with higher specificity to fused genes during alignment³
This can be problematic with FFPE samples that generally contain fragmented DNA/RNA³

Filtering of reads

Reads originating from highly expressed genes that are unlikely to be involved in gene fusions, such as ribosomal RNA, must be anticipated and filtered out appropriately³

Reads ratio

Low ratio of chimeric to wild type reads in the vicinity of the fusion boundary may indicate spurious mismapping of reads³

How to detect gene fusions

Highly promising fusion-sequence predictions evident if sequences in the read strongly align to sequences in 1 of the genes that constitute the gene fusion³
Gene fusions usually follow splicing patterns of wild-type genes; thus, their detection at exon boundaries that match those of the wild-type gene(s) is another powerful indicator of gene fusion sequence identification³

PLANNING FOR YOUR PRACTICE

Chosen library type should be supportive of laboratory sample batching considerations and be able to comprehensively detect gene fusions (known/novel)3

Review batching considerations across all testing methodologies

NGS test reporting

A clear and concise report is crucial to ensure immediate clinical use to the ordering physician4

The report should contain all the information required for the ordering clinician to know what was tested and the results obtained from the test. Incomplete or unclear representation of the data can lead to clinical errors and incorrect patient management.4

All clinically critical information should be clear and concise and displayed prominently at the beginning of the report, to increase the likelihood that it will be seen and understood
Using graphs, charts, and tables may help increase clarity of the report4
Recommendations should be made where possible when based on clinical evidence and appropriately cited4
Reports should not be limited to positive findings. Pertinent negatives are equally important in clinical decisions and, thus, should also be reported4
Report any additional preanalytical, analytical, or postanalytical testing variables, as these can provide context for some test results and influence the result’s clinical interpretation4

In addition, details about the testing methodology should be presented at the bottom of the report, including description of the method, assay performance characteristics, and critical quality metrics for the assay run. Unless the entire gene is tested, specific gene loci, exons, or hot spots tested should be listed in the final report.⁴

Review a sample test report5

Detected genomic alterations should be classified under a 4-tiered system4
Tier I	Variants of strong clinical significance Therapies approved by the FDA, and professional guidelines, or on well-powered clinical studies with expert consensus Diagnostic or prognostic biomarker in specific tumor types, based on professional guidelines or well-powered clinical studies with expert consensus
Tier II	Variants of potential clinical significance Therapies based on FDA-approved therapies for different tumor types or investigational therapies Based on multiple small studies with some consensus Preclinical trials or a few case reports without consensus
Tier III	Variants of unknown clinical significance None or no convincing evidence to determine clinical/biological significance
Tier IV	Variants of known insignificance No existing published evidence of cancer association Observed at significant allele frequencies in the general population or a specific subpopulation

DETECTED GENOMIC ALTERATIONS SHOULD BE CLASSIFIED UNDER A 4-TIERED SYSTEM4
Tier I	Variants of strong clinical significance Therapies approved by the FDA, and professional guidelines, or on well-powered clinical studies with expert consensus Diagnostic or prognostic biomarker in specific tumor types, based on professional guidelines or well-powered clinical studies with expert consensus
Tier II	Variants of potential clinical significance Therapies based on FDA-approved therapies for different tumor types or investigational therapies Based on multiple small studies with some consensus Preclinical trials or a few case reports without consensus
Tier III	Variants of unknown clinical significance None or no convincing evidence to determine clinical/biological significance
Tier IV	Variants of known insignificance No existing published evidence of cancer association Observed at significant allele frequencies in the general population or a specific subpopulation

Tier I–III alterations must be reported in descending order of clinical importance4
Clinically useful interpretations should be provided for tier I and II variants to inform disease management decisions4
Interpretations for tier III variants should be brief, bearing in mind the goal to keep critical information clear and concise4
Tier IV alterations are benign or likely benign; therefore, it is recommended that they not be reported4

Treatment decisions are multifaceted and often based on several factors that are unknown to the molecular pathologist. Therefore, recommendations in an NGS test report should be relevant to the patient’s cancer diagnosis and contain language clarifying that the recommendations are based on data available to the laboratory, but that additional factors need to be considered to create a treatment plan for each individual.⁴

Nomenclature

Detected genomic alterations should be annotated and reported by the HUGO Gene Nomenclature Committee. Gene fusions should be reported listing both gene partners separated by a dash. For example, a gene fusion between the NTRK1 gene and the partner gene TPM3 should be reported as TPM3-NTRK1 4

Use of standard nomenclature does not outweigh the goal of clear communication. Use colloquial nomenclature in addition to standard nomenclature, as needed, to clearly communicate results to the clinical provider.⁴

Other reporting elements

The report should also contain any information that can help provide a comparison with other results from the patient over time or provide additional context for the reported results, including4

Genomic coordinates
Genome build
Transcript reference sequence
Pertinent negatives
Sequencing coverage cutoff for the NGS assay used
Genes and/or hotspots that did not meet the sequencing coverage criteria should be declared as failed

Role of the Multidisciplinary Molecular Tumor Board

With rapid advancements in sequencing technology, implementing multidisciplinary molecular tumor board may be crucial to help translate genomic alteration findings into evidence-based recommendations.⁶ Diverse perspectives provide a multidisciplinary evaluation of the patient, as well as ensure expert interpretation of complex genomic data, especially when using whole-exome or whole-genome sequencing.^6,7 Thus, the molecular tumor board should invite collaboration from multiple disciplines, including clinicians with varying specialties (eg, oncologists, hematologists, pathologists, geneticists, molecular biologists, and bioinformaticians).6,7

Molecular tumor boards can provide education, facilitate interpretation, and implementation of precision medicine by 6

Improving clinician’s understanding of assay strengths, limitations, and results
Increasing clinician’s confidence in and efficiency of utilizing molecular diagnostics
Providing information about laboratory updates, analysis software, and challenges in data interpretation and utilization

PLANNING FOR YOUR PRACTICE

Clear and actionable communication is paramount when reporting NGS results to the clinical provider4

cDNA, complementary DNA; DNA, deoxyribonucleic acid; FDA, US Food and Drug Administration; FFPE, formalin-fixed paraffin-embedded; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; RNA, ribonucleic acid.

References:

Aziz N, Zhao Q, Bry L, et al. College of American Pathologists’ laboratory standards for next-generation sequencing clinical tests. Arch Pathol Lab Med. 2015;139(4):481-493. Return to content
Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. Return to content
Conesa A, Madrigal P, Tarazona S, et al. A survey of best practices for RNA-seq data analysis. Genome Biol. 2016;17(13):1-19. Return to content
Li MM, Datto M, Duncavage EJ, et al. Standards and guidelines for the interpretation and reporting of sequence variants in cancer: a joint consensus recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists. J Mol Diagn. 2017;19(1):4-23. Return to content
Amatu A, Sartore-Bianchi A, Siena S. ESMO Open. 2016;1:e000023. Return to content
van der Velden DL, van Herpen CML, van Laarhoven HWM, et al. Molecular Tumor Boards: current practice and future needs. Ann Oncol. 2017;28(12):3070-3075. Return to content
Knepper TC, Bell GC, Hicks JK, et al. Key lessons learned from Moffitt’s molecular tumor board: the Clinical Genomics Action Committee experience. Oncologist. 2017;22(2):144-151. Return to content

Choosing a Commercial NGS Kit

Choosing a Commercial NGS Kit

A variety of factors need to be considered when deciding which commercial kit should be implemented within a lab setting

Assay considerations to support the decision-making process when choosing a commercial kit

Platform availability and cost¹
Manufacturer technical support¹
Assay requirements, ie, number of genes and the extent of gene coverage¹

Laboratory-specific considerations to ensure implementation of efficient workflows

In-house technical expertise¹
Expected testing volume and scalability¹
Required result turnaround time¹

Recognize potential issues that may affect turnaround time

Commercially available NGS kits2-26

VENDOR	KIT NAME	SAMPLE	# OF GENES INCLUDED	LIBRARY PREP METHOD	DNA/RNA	NTRK 1, 2, AND 3	UNKNOWN FUSION PARTNERS	INTEGRATED BIOINFORMATICS	RECOMMENDED SEQUENCER
Illumina^®	TruSight™ Oncology 500	FFPE	523	Hybrid Capture	DNA/RNA	Y	Y	Variant Calling + Interpretation/ Reporting (powered by PierianDx)	NextSeq^®
	TruSight^® Tumor 170	FFPE	170	Hybrid Capture	DNA/RNA	Y	Y	Variant Calling + Interpretation/ Reporting (powered by PierianDx)	NextSeq^®/ HiSeq
	TruSight™ RNA Fusion Panel	FFPE, bone marrow	507	Hybrid Capture	RNA	Y	Y	Variant Calling	MiniSeq™/ MiSeq™/ NextSeq^®
	TruSight^® RNA Pan-Cancer Panel	FFPE	1385	Hybrid Capture	RNA	Y	Y	Variant Calling	MiniSeq™/ MiSeq™/ NextSeq^®
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	FFPE	52	Amplicon	DNA/RNA	Y	N	Ion Reporter/ Oncomine™ Report	Ion PGM^® Dx System
	Ion Torrent™ Oncomine™ Comprehensive Assay v3	FFPE	161	Amplicon	DNA/RNA	Y	N	Ion Reporter/ Oncomine™ Report	Ion Chef™ and Ion S5™ Systems
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	FFPE, blood and bone marrow	203	Amplicon	DNA/RNA	Y	N	Ion Reporter/ Oncomine™ Report	Ion GeneStudio™ S5/Ion Chef™
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	Fresh frozen or FFPE	53	Anchored Multiplex PCR	RNA	Y	Y	Archer Analysis	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® Oncology Research Kit	Fresh frozen or FFPE	74	Anchored Multiplex PCR	RNA	Y	Y	Archer Analysis	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® Sarcoma Kit	Fresh frozen or FFPE	26	Anchored Multiplex PCR	RNA	*NTRK3* Only	Y	Archer Analysis	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® CTL Kit	Fresh frozen or FFPE	36	Anchored Multiplex PCR	RNA	Y	Y	Archer Analysis	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® Lung Kit	Fresh frozen or FFPE	14	Anchored Multiplex PCR	RNA	Y	Y	Archer Analysis	Illumina^®/ Ion Torrent™
Qiagen	GeneRead QIAact Lung DNA UMI Panel	FFPE/ liquid biopsy	19	UMI/PCR	DNA	*NTRK1* Only	N	Qiagen Clinical Insight	GeneReader
	GeneRead QIAact Lung RNA Fusion UMI Panel	FFPE/ liquid biopsy	19	UMI/PCR	DNA	*NTRK1* Only	N	Qiagen Clinical Insight	GeneReader

VENDOR	KIT NAME	SAMPLE
Illumina^®	TruSight™ Oncology 500	FFPE
	TruSight^® Tumor 170	FFPE
	TruSight™ RNA Fusion Panel	FFPE, bone marrow
	TruSight^® RNA Pan-Cancer Panel	FFPE
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	FFPE
	on Torrent™ Oncomine™ Comprehensive Assay v3	FFPE
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	FFPE, blood and bone marrow
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	Fresh frozen or FFPE
	Archer^® FusionPlex^® Oncology Research Kit	Fresh frozen or FFPE
	Archer^® FusionPlex^® Sarcoma Kit	Fresh frozen or FFPE
	Archer^® FusionPlex^® CTL Kit	Fresh frozen or FFPE
	Archer^® FusionPlex^® Lung Kit	Fresh frozen or FFPE
Qiagen	GeneRead QIAact Lung DNA UMI Panel	FFPE/ liquid biopsy
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	FFPE/ liquid biopsy

VENDOR	KIT NAME	# OF GENES INCLUDED
Illumina^®	TruSight™ Oncology 500	523
	TruSight^® Tumor 170	170
	TruSight™ RNA Fusion Panel	507
	TruSight^® RNA Pan-Cancer Panel	1385
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	52
	on Torrent™ Oncomine™ Comprehensive Assay v3	161
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	203
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	53
	Archer^® FusionPlex^® Oncology Research Kit	74
	Archer^® FusionPlex^® Sarcoma Kit	26
	Archer^® FusionPlex^® CTL Kit	36
	Archer^® FusionPlex^® Lung Kit	14
Qiagen	GeneRead QIAact Lung DNA UMI Panel	19
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	19

VENDOR	KIT NAME	LIBRARY PREP METHOD
Illumina^®	TruSight™ Oncology 500	Hybrid Capture
	TruSight^® Tumor 170	Hybrid Capture
	TruSight™ RNA Fusion Panel	Hybrid Capture
	TruSight^® RNA Pan-Cancer Panel	Hybrid Capture
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	Amplicon
	on Torrent™ Oncomine™ Comprehensive Assay v3	Amplicon
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	Amplicon
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	Anchored Multiplex PCR
	Archer^® FusionPlex^® Oncology Research Kit	Anchored Multiplex PCR
	Archer^® FusionPlex^® Sarcoma Kit	Anchored Multiplex PCR
	Archer^® FusionPlex^® CTL Kit	Anchored Multiplex PCR
	Archer^® FusionPlex^® Lung Kit	Anchored Multiplex PCR
Qiagen	GeneRead QIAact Lung DNA UMI Panel	UMI/PCR
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	UMI/PCR

VENDOR	KIT NAME	DNA/RNA
Illumina^®	TruSight™ Oncology 500	DNA/RNA
	TruSight^® Tumor 170	DNA/RNA
	TruSight™ RNA Fusion Panel	RNA
	TruSight^® RNA Pan-Cancer Panel	RNA
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	DNA/RNA
	on Torrent™ Oncomine™ Comprehensive Assay v3	DNA/RNA
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	DNA/RNA
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	RNA
	Archer^® FusionPlex^® Oncology Research Kit	RNA
	Archer^® FusionPlex^® Sarcoma Kit	RNA
	Archer^® FusionPlex^® CTL Kit	RNA
	Archer^® FusionPlex^® Lung Kit	RNA
Qiagen	GeneRead QIAact Lung DNA UMI Panel	DNA
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	DNA

VENDOR	KIT NAME	NTRK 1, 2, AND 3
Illumina^®	TruSight™ Oncology 500	Y
	TruSight^® Tumor 170	Y
	TruSight™ RNA Fusion Panel	Y
	TruSight^® RNA Pan-Cancer Panel	Y
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	Y
	on Torrent™ Oncomine™ Comprehensive Assay v3	Y
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	Y
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	Y
	Archer^® FusionPlex^® Oncology Research Kit	Y
	Archer^® FusionPlex^® Sarcoma Kit	*NTRK3* Only
	Archer^® FusionPlex^® CTL Kit	Y
	Archer^® FusionPlex^® Lung Kit	Y
Qiagen	GeneRead QIAact Lung DNA UMI Panel	*NTRK1* Only
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	*NTRK1* Only

VENDOR	KIT NAME	UNKNOWN FUSION PARTNERS
Illumina^®	TruSight™ Oncology 500	Y
	TruSight^® Tumor 170	Y
	TruSight™ RNA Fusion Panel	Y
	TruSight^® RNA Pan-Cancer Panel	Y
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	N
	on Torrent™ Oncomine™ Comprehensive Assay v3	N
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	N
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	Y
	Archer^® FusionPlex^® Oncology Research Kit	Y
	Archer^® FusionPlex^® Sarcoma Kit	Y
	Archer^® FusionPlex^® CTL Kit	Y
	Archer^® FusionPlex^® Lung Kit	Y
Qiagen	GeneRead QIAact Lung DNA UMI Panel	N
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	N

VENDOR	KIT NAME	INTEGRATED BIOINFORMATICS
Illumina^®	TruSight™ Oncology 500	Variant Calling + Interpretation/ Reporting (powered by PierianDx)
	TruSight^® Tumor 170	Variant Calling + Interpretation/ Reporting (powered by PierianDx)
	TruSight™ RNA Fusion Panel	Variant Calling
	TruSight^® RNA Pan-Cancer Panel	Variant Calling
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	Ion Reporter/ Oncomine™ Report
	on Torrent™ Oncomine™ Comprehensive Assay v3	Ion Reporter/ Oncomine™ Report
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	Ion Reporter/ Oncomine™ Report
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	Archer Analysis
	Archer^® FusionPlex^® Oncology Research Kit	Archer Analysis
	Archer^® FusionPlex^® Sarcoma Kit	Archer Analysis
	Archer^® FusionPlex^® CTL Kit	Archer Analysis
	Archer^® FusionPlex^® Lung Kit	Archer Analysis
Qiagen	GeneRead QIAact Lung DNA UMI Panel	Qiagen Clinical Insight
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	Qiagen Clinical Insight

VENDOR	KIT NAME	RECOMMENDED SEQUENCER
Illumina^®	TruSight™ Oncology 500	NextSeq^®
	TruSight^® Tumor 170	NextSeq^®/ HiSeq
	TruSight™ RNA Fusion Panel	MiniSeq™/ MiSeq™/ NextSeq^®
	TruSight^® RNA Pan-Cancer Panel	MiniSeq™/ MiSeq™/ NextSeq^®
Thermo Fisher	Ion Torrent™ Oncomine™ Focus Assay	Ion PGM^® Dx System
	on Torrent™ Oncomine™ Comprehensive Assay v3	Ion Chef™ and Ion S5™ Systems
	Ion Torrent™ Oncomine™ Childhood Cancer Research Assay	Ion Chef™ and Ion S5™ Systems
Archer DX	Archer^® FusionPlex^® Solid Tumor Kit	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® Oncology Research Kit	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® Sarcoma Kit	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® CTL Kit	Illumina^®/ Ion Torrent™
	Archer^® FusionPlex^® Lung Kit	Illumina^®/ Ion Torrent™
Qiagen	GeneRead QIAact Lung DNA UMI Panel	GeneReader
Qiagen	GeneRead QIAact Lung RNA Fusion UMI Panel	GeneReader

Information current as of February 2019.
Contact the laboratory vendors for more information. This list may not represent all tests for NTRK1, NTRK2, and NTRK3 gene fusions.
All kits listed above are for research use only (RUO).

Review important considerations for selecting a commercial NGS kit with Dr Jessica Davis

DNA, deoxyribonucleic acid; FFPE, formalin-fixed paraffin-embedded; NGS, next-generation sequencing; RNA, ribonucleic acid; NTRK, neurotrophic tyrosine receptor kinase.

References:

Jennings LJ, Arcila ME, Croless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. Return to content
Illumina. TruSight Oncology 500 DNA/RNA Kit. https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500.html. Accessed February 27, 2019. Return to content
Illumina. TruSight Tumor 170 Datasheet. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/trusight-tumor-170-data-sheet-1170-2016-017.pdf. Accessed February 27, 2019. Return to content
Illumina. TruSight RNA fusion panel. https://www.illumina.com/products/by-type/clinical-research-products/trusight-rna-pan-cancer.html. Accessed February 27, 2019. Return to content
Illumina. NextSeq 550 data sheet. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/nextseq-550-system-spec-sheet-770-2013-053.pdf. Accessed March 1, 2019. Return to content
Illumina. TruSight RNA Pan-Cancer Panel. https://www.illumina.com/products/by-type/clinical-research-products/trusight-rna-pan-cancer.html. Accessed March 1, 2019. Return to content
Illumina. TruSight RNA Pan-Cancer Panel Datasheet. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/trusight-rna-pan-cancer-data-sheet-1170-2015-004.pdf. Accessed February 28, 2019. Return to content
lllumina. TruSight RNA Pan-Cancer Sequencing Panel Gene List. https://www.illumina.com/content/dam/illumina-marketing/documents/products/gene_lists/gene_list_trusight_pan_cancer.xlsx. Accessed February 27, 2019. Return to content
ThermoFisher. Oncomine Focus Assay, 318 Solution. https://www.thermofisher.com/order/catalog/product/A28548. Accessed February 28, 2019. Return to content
ThermoFisher. Oncomine Focus Assay White Paper. https://tools.thermofisher.com/content/sfs/brochures/oncomine-focus-assay-performance-white-paper.pdf. Accessed February 28, 2019. Return to content
ThermoFisher. Oncomine Comprehensive Assay v3 flyer. https://assets.thermofisher.com/TFS-Assets/LSG/brochures/oncomine-comprehensive-assay-v3-flyer.pdf. Accessed February 28, 2019. Return to content
ThermoFisher. Oncomine Childhood Cancer Research Assay. https://www.thermofisher.com/us/en/home/clinical/preclinical-companion-diagnostic-development/oncomine-oncology/oncomine-childhood-research-assay.html. Accessed February 27, 2019. Return to content
ThermoFisher Cancer Genomics Research Brochure. https://tools.thermofisher.com/content/sfs/brochures/cancer-genomics-research-brochure.pdf. Accessed February 27, 2019. Return to content
ArcherDX. Archer FusionPlex NGS Assays. http://archerdx.com/fusionplex-assays/. Accessed February 28, 2019. Return to content
ArcherDX. Archer FusionPlex Solid Tumor Kit. http://archerdx.com/fusionplex-assays/solid-tumor. Accessed February 28, 2019. Return to content
ArcherDX. Archer FusionPlex Solid Tumor Kit Product Insert. http://info.archerdx.com/acton/attachment/17223/f-05ae/1/-/-/l-0014/l-0014:2353/LA179-Product-Insert-FusionPlex-Solid-Tumor.pdf?nc=1. Accessed March 2, 2019. Return to content
ArcherDX. Archer FusionPlex Oncology Research Kit. http://archerdx.com/fusionplex-assays/oncology-research. Accessed March 4, 2019. Return to content
ArcherDx. Archer FusionPlex Oncology Research Kit Product Insert. http://info.archerdx.com/acton/attachment/17223/f-05b0/1/-/-/l-0014/l-0014:2356/LA181-Product-Insert-FusionPlex-Oncology-Research.pdf?nc=1. Accessed February 28, 2019. Return to content
Archer FusionPlex Sarcoma Kit. https://archerdx.com/fusionplex-assays/sarcoma. Accessed March 4, 2019. Return to content
Archer FusionPlex Sarcoma Kit Product insert. http://cdn2.hubspot.net/hubfs/4445440/Product%20inserts/LA168.B%20Product%20Insert,%20FusionPlex%20Sarcoma.pdf. Accessed February 28, 2019. Return to content
Archer FusionPlex CTL Kit. https://archerdx.com/fusionplex-assays/ctl-rna. Accessed February 28, 2019. Return to content
Archer FusionPlex CTL Kit. Product Insert. http://cdn2.hubspot.net/hubfs/4445440/Product%20inserts/LA180.B%20Product%20Insert,%20FusionPlex%20CTL.pdf. Accessed February 28, 2019. Return to content
Archer FusionPlex Lung Kit. Product Insert. http://cdn2.hubspot.net/hubfs/4445440/Product%20inserts/LA671.B%20Product%20Insert,%20FusionPlex%C2%AE%20Lung,%20SK0133.pdf. Accessed March 2, 2019. Return to content
Archer FusionPlex Lung Kit. Protocol. http://cdn2.hubspot.net/hubfs/4445440/Protocols/LA135.F%20Protocol,% 20Archer%20FusionPlex%20Kits%20for%20Illumina.pdf. Accessed March 2, 2019. Return to content
Qiagen. GeneRead QIAact Lung DNA UMI Panel Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=94ab92d2-1918-4388-989b-4cefa8eed203&lang=en. Accessed March 4, 2019. Return to content
Qiagen. GeneRead QIAact Lung RNA Fusion UMI Panel Handbook. https://www.qiagen.com/us/resources/resourcedetail?id=1a71d98a-c45c-44fa-b4af-874cd1d2b61f&lang=en. Accessed March 4, 2019. Return to content

Reference NGS Laboratories

Reference NGS Laboratories

Commercial laboratories currently offering gene fusion testing inclusive of NTRK1, NTRK2, and NTRK3

NantHealth

NAVICAN

Neogenomics Laboratories

Paradigm Diagnostics

PathGroup

Tempus

NTRK, neurotrophic tyrosine receptor kinase.