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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Abstract

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

Introduction

The fusion of two or more genes into a single transcriptional entity can occur as the result of large scale chromosomal variations including deletions, duplications, insertions, inversions, and translocations. Through altered transcriptional control and/or altered functional properties of the expressed gene product, these fusion genes can confer oncogenic properties to cancer cells1. In many cases, fusion genes are known to act as primary oncogenic drivers by directly activating cellular proliferation and survival pathways.

The clinical relevance of gene fusions for cancer patients first became apparent with the disc....

Protocol

1. Library preparation and sequencing

  1. General assay considerations and pre-assay steps
    1. Assay runs typically consist of seven clinical samples and one positive control (although the number of samples per library preparation run can be adjusted as necessary). Use a positive control that contains at least several gene fusions (that the assay targets) that have been confirmed by a manufacturer and/or have been confirmed by an orthogonal methodology. A non-template control (NTC) must b.......

Representative Results

Shown in Figure 3, Figure 4 and Figure 5 are screenshots from the analysis user interface demonstrating results from a lung adenocarcinoma sample. In Figure 3, the sample summary is shown (top) that lists the called strong evidence fusions, as well as the QC status (circled in red). The ADCK4-NUMBL fusion (of which 3 isoforms are listed) is immediately ignored because it is a persistent transcr.......

Discussion

Anchored multiplex PCR-based target enrichment and library preparation followed by next-generation sequencing is well suited for multiplexed gene fusion assessment in clinical tumor samples. By focusing on RNA input rather than genomic DNA, the need to sequence large and repetitive introns is avoided. Additionally, since this approach amplifies gene fusions regardless of the identity of the fusion partner, novel fusions are detected. This is a critical advantage in the clinical realm, and there have been many examples of.......

Acknowledgements

This work was supported by the Molecular Pathology Shared Resource of the University of Colorado (National Cancer Institute Cancer Center Support Grant No. P30-CA046934) and by the Colorado Center for Personalized Medicine.

....

Materials

NameCompanyCatalog NumberComments
10 mM Tris HCl pH 8.0IDT11-05-01-13Used for TNA dilution
1M Tris pH 7.0Thermo FisherAM9850GUsed in library pooling
25 mL Reagent Reservoir with dividerUSA Scientific9173-2000For use with multi-channel pipetters and large reagent volumes
96-well TemPlate Semi-Skirt 0.1mL PCR plate-naturalUSA Scientific1402-9700Plate used for thermocycler steps
Agencourt AMPure XP BeadsBeckman CoulterA63881Used in purification after several assay steps
Agencourt Formapure KitBeckman CoulterA33343Used in TNA extraction
Archer FusionPlex Solid Tumor kitArcherDXAB0005This kit contains most of the reagents necessary to perform library preperation for Illumina sequencing (kits for Ion Torrent sequencing are also available)
Cold block, 96-wellLight LabsA7079Used for keeping samples chilled at various steps
EthanolDecon LabsDSP-MD.43Used for bead washes
Library Quantification for Illumina Internal Control StandardKapa BiosystemsKK4906Used for library quantitation
Library Quantification Primers and ROX Low qPCR mixKapa BiosystemsKK4973Used for library quantitation
Library Quantification StandardsKapa BiosystemsKK4903Used for library quantitation
Magnet Plate, 96-well (N38 grade)AlpaquaA32782Used in bead purificiation steps
MBC Adapters Set BArcherDXAK0016-48Adapters that contain sample-specific indexes to enable multiplex sequencing
Micro CentrifugeUSA Scientific2641-0016Used for spinning down PCR tubes
MicroAmp EnduraPlate Optical 96 well PlateThermo-Fisher4483485Used for Pre-Seq QClibrary quantitation
Microamp Optical Film Compression PadApplied Biosystems4312639Used for library quantitation
Mini Plate SpinnerLabnetMPS-1000Used for collecing liquid at bottom of plate wells
MiSeq Reagent Kit v3 (600 cycle)IlluminaMS-102-3003Contains components necessary for a MiSeq sequencing run
MiSeqDx SystemIlluminaNGS Sequencing Instrument
Model 9700 ThermocyclerApplied BiosystemsUsed for several steps during assay
nuclease free waterAmbion9938Used as general diluent
Optical ABI 96-well PCR plate coversThermo-Fisher4311971Used for Pre-Seq QClibrary quantitation
PCR Workstation Model 600Air Clean SystemsBZ10119636Wet-bench assay steps performed in this 'dead air box'
Proteinase KQiagen1019499Used in TNA extraction
QuantStudio 5Applied BiosystemsLSA28139qPCR instrument used for PreSeq and library quantitation
Qubit RNA HS Assay KitLife TechnologiesQ32855Use for determing RNA concentration in TNA samples
RNase AwayFisher12-402-178Used for general RNase decontamination of work areas
Seraseq FFPE Tumor Fusion RNA Reference Material v2SeraCare0710-0129Used as the assay positive control
Sodium HydroxideFisherBP359-212Used in clean-up steps and for sequencing setup
SYBR Green SupermixBio Rad172-5120Component of PreSeq QC Assay
TempAssure PCR 8-tube StripsUSA Scientific1402-2700Used for reagent and sample mixing etc.
Template RT PCR filmUSA Scientific2921-7800Used for covering 96-well plates
U-Bottom 96-well MicroplateLSPMP8117-RUsed during bead purification

References

  1. Mitelman, F., Johansson, B., Mertens, F. The impact of translocations and gene fusions on cancer causation. Nature Reviews Cancer. 7 (4), 233-245 (2007).
  2. Daley, G. Q., Van Etten, R. A., Baltimore, D.

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