Thermo Fisher Scientific • 200 Oyster Point Boulevard, South San Francisco, CA 94080• thermofisher.com Introduction Gene fusions play an important role in oncogenesis and the progression of cancer 1 . As important biomarkers, sensitive identification of gene fusions is critical to future oncology research. Next-generation sequencing (NGS) with Ion AmpliSeq TM targeted enrichment enables simple, accurate and specific detection of relevant fusion isoforms. Here we introduce a novel automatic high-multiplexing primer design strategy that has the flexibility to develop customized AmpliSeq fusion panels for any combination of fusion isoforms, scaling to panels that can detect thousands of isoforms in a single primer pool, which increases the sensitivity of fusion detection while decreasing the sample input required to as low as 10 ng. Methods A novel primer design pipeline was developed to support the design of custom AmpliSeq gene fusion panels. A minimum set of information is required for the precise design of amplicons targeting specific gene fusion breakpoints. However, this information is generalizable for all gene fusions where the breakpoint has been previously characterized. As summarized in Figure 1, for any given fusion isoform, amplicons are designed strategically to gene fusion junctions. The design input criteria for the automated primer design pipeline only includes the specific gene name, transcript identifier and exon level information. The Gene, Transcript and Exon information for targeted gene fusions is entered into a file that is used by the pipeline to generate the automated design. Every record in the file represents a unique RNA fusion isoform to establish an easy-parsing format for the pipeline. Specific gene, transcript and exon level information for every fusion design is required (Table 1). The pipeline locates the fusion breakpoint position, extracts gene sequences of every candidate fusion target and builds the fusion reference. Candidate amplicons are generated against the fusion reference. According to the design requirements of pool number and the conflicts among primer pairs, the pipeline performs pooling to minimize primer interactions. As the examples shown in Figure 2, there could be different fusions involving the specified pair of genes derived from a number of different breakpoints. The pipeline has the capability to cover different fusion targets in a single panel based on design requirements. Finally, the pipeline generates an optimal set of amplicons for targeted isoforms in one AmpliSeq fusion assay. The assay is based on Ion AmpliSeq technology and is designed for use with the Ion Torrent™ sequencing platforms (Figure 3). Conclusions In summary, we have developed an automatic pipeline that can generate robust, comprehensive customized multiplex RNA fusion assays for targeted next-generation sequencing. References 1. Gao, Q., Liang, W.W., Foltz, S.M., Mutharasu, G., Jayasinghe, R.G., Cao, S., Liao, W.W., Reynolds, S.M., Wyczalkowski, M.A., Yao, L. and Yu, L., 2018. Driver fusions and their implications in the development and treatment of human cancers. Cell reports, 23(1), pp.227-238. Acknowledgements R&D team and Product Management team, South San Francisco, CA R&D team, Carlsbad, CA R&D team, Ann Arbor, MI Posters from scientists at Thermo Fisher Scientific: www.thermofisher.com/2020AACR-posters Contact information: [email protected] . Na Li 1 , Antonio Martinez Alcantara 1 , Aren Ewing 2 , Rajesh K Gottimukkala 1 , Fiona Hyland 1 , Seth Sadis 3 , (1)Thermo Fisher, South San Francisco, CA, 94080, (2)Thermo Fisher, Carlsbad, CA, 92008, (3)Thermo Fisher, Ann Arbor, MI 48104 Development of customizable targeted RNA fusion panels using a novel automated high- multiplexing primer design strategy Figure 3. Ion Torrent comprehensive NGS workflow Gene A Gene B Fusion gene Fusion transcript Fusion Junction Amplicon Translocation Transcription 5’ 3’ 5’ 3’ Figure 1. Targeted fusion junction assay design Figure 2. Example of different type of fusion targets Figure 4. Customized AmpliSeq and AmpliSeq HD fusion panel on ampliseq.com Genexus TM system GeneStudio TM S5 system Item Description Gene name The symbol of the Gene A and Gene B involved in the fusion Transcript ID ENSEMBL identifier for the Gene A and Gene B Exon number Exon number where breakpoint is present. Breakpoint position Breakpoint position Table 1. Design required information This pipeline has been used to design the fusion assays contained in Oncomine TM Focus and Comprehensive assays, Oncomine Precision Assay, and others. Oncomine Precision Assay was tested on the Ion Torrent Genexus integrated sequencer. Testing on FFPE samples with known positive fusions confirms that the expected fusions were detected with 100% accuracy. Custom fusion panels are available on Ion AmpliSeq Designer (ampliseq.com) , a cloud based software enables users to create highly multiplexed NGS panels. High quality fusion amplicons that are available on ampliseq.com were pre-designed by this pipeline. Users could search and select targeted gene symbols and the available fusion partners to create custom fusion panel efficiently. For further requirements, customers can use AmpliSeq White Glove service for designing highly customized fusion panels. Fusion RNA Primary Cancer Tissue Detected fusion isoform 10% dilution Read counts, r1* 10% dilution Read counts, r2* EML4-ALK Lung EML4-ALK.E13A20.COSF408.2 578 591 KIF5B-RET Lung KIF5B-RET.K24R11.COSF1262.1 571 675 NCOA4-RET Thyroid NCOA4-RET.N7R12.COSF1491.1 881 978 CD74-ROS1 Lung CD74-ROS1.C6R34.COSF1200.1 95 400 SLC34A2-ROS1 Lung, Stomach SLC34A2-ROS1.S4R34.COSF1198 234 58 TPM3-NTRK1 Lung, Intestine TPM3-NTRK1.T7N10.COSF1329 1043 1125 FGFR3-BAIAP2L1 Urinary tract FGFR3-BAIAP2L1.F17B2.COSF1346 657 921 FGFR3-TACC3 Urinary tract, CNS FGFR3-TACC3.F17T11.COSF1348.1 1116 1311 ETV6-NTRK3 Multiple ETV6-NTRK3.E5N15.COSF571.2 547 960 LMNA-NTRK1 Skin LMNA-NTRK1.L2N11 1102 908 SLC45A3-BRAF Prostate SLC45A3-BRAF.S1B8.COSF871 437 452 MET exon14 skip Lung MET-MET.M13M15.1 513 342 EGFRvIII Brain EGFRvIII.E1E8.Del1 677 731 ETV6-NTRK2 ETV6-NTRK2.E5N16 0 0 KLK2-FGFR2 KLK2-FGFR2.K1F5 0 0 TFG-ALK TFG-ALK.T5A20.COSF426 0 0 Table 2. Read counts, Seraseq TM FFPE Tumor Fusion RNA Reference Material v2, 10% dilution into FFPE sample Result This pipeline has been used extensively to develop high performing multiplex customized AmpliSeq RNA fusion panels as well as AmpliSeq TM HD fusion panels with ultrahigh sensitivity for applications including liquid biopsy. The pipeline generates 175-base amplicons for use on formalin-fixed, paraffin-embedded (FFPE) samples or 120-base amplicons for use on cfRNA from blood samples. A single panel can include thousands of known fusion variants. Grey rows indicate negative controls: fusion isoforms not present in the control sample. *Sequencing experiments are performed in two independent occasions. 5’ partner 3’ partner Detected fusion isoform 10% dilution Read counts, r1* 10% dilution Read counts, r2* TPM3 NTRK1 TPM3-NTRK1.T7N10.COSF1329 421 464 LMNA NTRK1 LMNA-NTRK1.L11N11.1 366 308 IRF2BP2 NTRK1 IRF2BP2-NTRK1.I1N10 151 218 SQSTM1 NTRK1 SQSTM1-NTRK1.S5N10.1 493 316 TFG NTRK1 TFG-NTRK1.T5N10.COSF1328 276 280 AFAP1 NTRK2 AFAP1-NTRK2.A14N12 381 456 NACC2 NTRK2 NACC2-NTRK2.N4N13.COSF1448 198 468 QKI NTRK2 QKI-NTRK2.Q6N16.COSF1446 418 689 TRIM24 NTRK2 TRIM24-NTRK2.T12N15.1 184 362 PAN3 NTRK2 PAN3-NTRK2.P1N17 82 99 ETV6 NTRK3 ETV6-NTRK3.E4N14.1 400 663 ETV6 NTRK3 ETV6-NTRK3.E4N15.COSF823.2 400 330 ETV6 NTRK3 ETV6-NTRK3.E5N14.1 305 432 ETV6 NTRK3 ETV6-NTRK3.E5N15.COSF571.2 328 357 BTBD1 NTRK3 BTBD1-NTRK3.B4N14 217 433 EML4-ALK.E6aA20.AB374361.1 0 0 CD74-ROS1.C6R34.COSF1200.1 0 0 KIF5B-RET.K24R11.COSF1262.1 0 0 Table 3. Read counts, Seraseq TM FFPE NTRK Fusion RNA Reference Material, 10% dilution into FFPE sample Grey rows indicate negative controls: fusion isoforms not present in the control sample *Sequencing experiments are performed in two independent occasions. For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. SERASEQ is a trademark of SeraCare Life Sciences, Inc. Amplicons are designed strategically targeted for fusion junction. The assay is designed for use with the Ion Torrent sequencing platforms with comprehensive end-to-end NGS research workflow. Gene B Gene A 5’ 3’ 5’ 3’
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Thermo Fisher Scientific • 200 Oyster Point Boulevard, South San Francisco, CA 94080• thermofisher.com
IntroductionGene fusions play an important role in oncogenesis and the progression of cancer1. As important biomarkers, sensitive identification of gene fusions is critical to future oncology research. Next-generation sequencing (NGS) with Ion AmpliSeqTM targeted enrichment enables simple, accurate and specific detection of relevant fusion isoforms. Here we introduce a novel automatic high-multiplexing primer design strategy that has the flexibility to develop customized AmpliSeq fusion panels for any combination of fusion isoforms, scaling to panels that can detect thousands of isoforms in a single primer pool, which increases the sensitivity of fusion detection while decreasing the sample input required to as low as 10 ng.
MethodsA novel primer design pipeline was developed to support the design of custom AmpliSeq gene fusion panels. A minimum set of information is required for the precise design of amplicons targeting specific gene fusion breakpoints. However, this information is generalizable for all gene fusions where the breakpoint has been previously characterized. As summarized in Figure 1, for any given fusion isoform, amplicons are designed strategically to gene fusion junctions.
The design input criteria for the automated primer design pipeline only includes the specific gene name, transcript identifier and exon level information. The Gene, Transcript and Exon information for targeted gene fusions is entered into a file that is used by the pipeline to generate the automated design. Every record in the file represents a unique RNA fusion isoform to establish an easy-parsing format for the pipeline. Specific gene, transcript and exon level information for every fusion design is required (Table 1).
The pipeline locates the fusion breakpoint position, extracts gene sequences of every candidate fusion target and builds the fusion reference. Candidate amplicons are generated against the fusion reference. According to the design requirements of pool number and the conflicts among primer pairs, the pipeline performs pooling to minimize primer interactions. As the examples shown in Figure 2, there could be different fusions involving the specified pair of genes derived from a number of different breakpoints. The pipeline has the capability to cover different fusion targets in a single panel based on design requirements.
Finally, the pipeline generates an optimal set of amplicons for targeted isoforms in one AmpliSeq fusion assay. The assay is based on Ion AmpliSeq technology and is designed for use with the Ion Torrent™ sequencing platforms (Figure 3).
ConclusionsIn summary, we have developed an automatic pipeline that can generate robust, comprehensive customized multiplex RNA fusion assays for targeted next-generation sequencing.
References1. Gao, Q., Liang, W.W., Foltz, S.M., Mutharasu, G., Jayasinghe, R.G., Cao, S., Liao, W.W., Reynolds, S.M., Wyczalkowski, M.A., Yao, L. and Yu, L., 2018. Driver fusions and their implications in the development and treatment of human cancers. Cell reports, 23(1), pp.227-238.
AcknowledgementsR&D team and Product Management team, South San Francisco, CAR&D team, Carlsbad, CAR&D team, Ann Arbor, MI
Posters from scientists at Thermo Fisher Scientific: www.thermofisher.com/2020AACR-posters
Contact information: [email protected].
Na Li1, Antonio Martinez Alcantara1, Aren Ewing2, Rajesh K Gottimukkala1, Fiona Hyland1, Seth Sadis3, (1)Thermo Fisher, South San Francisco, CA, 94080, (2)Thermo Fisher, Carlsbad, CA, 92008, (3)Thermo Fisher, Ann Arbor, MI 48104
Development of customizable targeted RNA fusion panels using a novel automated high-multiplexing primer design strategy
Figure 3. Ion Torrent comprehensive NGS workflow
Gene A Gene B
Fusion gene
Fusion transcript
Fusion Junction
Amplicon
Translocation
Transcription
5’ 3’ 5’ 3’
Figure 1. Targeted fusion junction assay design
Figure 2. Example of different type of fusion targets
Figure 4. Customized AmpliSeq and AmpliSeq HD fusion panel on ampliseq.com
GenexusTM system GeneStudioTM S5 system
Item Description
Gene name The symbol of the Gene A and Gene B involved in the fusion
Transcript ID ENSEMBL identifier for the Gene A and Gene B
Exon number Exon number where breakpoint is present.
Breakpoint position Breakpoint position
Table 1. Design required information
This pipeline has been used to design the fusion assays contained in OncomineTM
Focus and Comprehensive assays, Oncomine Precision Assay, and others. OncominePrecision Assay was tested on the Ion Torrent Genexus integrated sequencer. Testing on FFPE samples with known positive fusions confirms that the expected fusions were detected with 100% accuracy.
Custom fusion panels are available on Ion AmpliSeq Designer (ampliseq.com), a cloud based software enables users to create highly multiplexed NGS panels. High quality fusion amplicons that are available on ampliseq.com were pre-designed by this pipeline. Users could search and select targeted gene symbols and the available fusion partners to create custom fusion panel efficiently. For further requirements, customers can use AmpliSeq White Glove service for designing highly customized fusion panels.
Fusion RNA Primary Cancer Tissue Detected fusion isoform 10% dilution
Read counts, r1*10% dilution Read counts, r2*
EML4-ALK Lung EML4-ALK.E13A20.COSF408.2 578 591
KIF5B-RET Lung KIF5B-RET.K24R11.COSF1262.1 571 675
NCOA4-RET Thyroid NCOA4-RET.N7R12.COSF1491.1 881 978
CD74-ROS1 Lung CD74-ROS1.C6R34.COSF1200.1 95 400
SLC34A2-ROS1 Lung, Stomach SLC34A2-ROS1.S4R34.COSF1198 234 58
TPM3-NTRK1 Lung, Intestine TPM3-NTRK1.T7N10.COSF1329 1043 1125
FGFR3-BAIAP2L1 Urinary tract FGFR3-BAIAP2L1.F17B2.COSF1346 657 921
FGFR3-TACC3 Urinary tract, CNS FGFR3-TACC3.F17T11.COSF1348.1 1116 1311
ETV6-NTRK3 Multiple ETV6-NTRK3.E5N15.COSF571.2 547 960
LMNA-NTRK1 Skin LMNA-NTRK1.L2N11 1102 908
SLC45A3-BRAF Prostate SLC45A3-BRAF.S1B8.COSF871 437 452
MET exon14 skip Lung MET-MET.M13M15.1 513 342
EGFRvIII Brain EGFRvIII.E1E8.Del1 677 731
ETV6-NTRK2 ETV6-NTRK2.E5N16 0 0
KLK2-FGFR2 KLK2-FGFR2.K1F5 0 0
TFG-ALK TFG-ALK.T5A20.COSF426 0 0
Table 2. Read counts, SeraseqTM FFPE Tumor Fusion RNA Reference Material v2, 10% dilution into FFPE sample
ResultThis pipeline has been used extensively to develop high performing multiplex customized AmpliSeq RNA fusion panels as well as AmpliSeqTM HD fusion panels with ultrahigh sensitivity for applications including liquid biopsy. The pipeline generates 175-base amplicons for use on formalin-fixed, paraffin-embedded (FFPE) samples or 120-base amplicons for use on cfRNA from blood samples. A single panel can include thousands of known fusion variants. Grey rows indicate negative controls: fusion isoforms not present in the control sample.
*Sequencing experiments are performed in two independent occasions.
5’ partner 3’ partner Detected fusion isoform 10% dilution Read counts, r1*
10% dilution Read counts, r2*
TPM3 NTRK1 TPM3-NTRK1.T7N10.COSF1329 421 464
LMNA NTRK1 LMNA-NTRK1.L11N11.1 366 308
IRF2BP2 NTRK1 IRF2BP2-NTRK1.I1N10 151 218
SQSTM1 NTRK1 SQSTM1-NTRK1.S5N10.1 493 316
TFG NTRK1 TFG-NTRK1.T5N10.COSF1328 276 280
AFAP1 NTRK2 AFAP1-NTRK2.A14N12 381 456
NACC2 NTRK2 NACC2-NTRK2.N4N13.COSF1448 198 468
QKI NTRK2 QKI-NTRK2.Q6N16.COSF1446 418 689
TRIM24 NTRK2 TRIM24-NTRK2.T12N15.1 184 362
PAN3 NTRK2 PAN3-NTRK2.P1N17 82 99
ETV6 NTRK3 ETV6-NTRK3.E4N14.1 400 663
ETV6 NTRK3 ETV6-NTRK3.E4N15.COSF823.2 400 330
ETV6 NTRK3 ETV6-NTRK3.E5N14.1 305 432
ETV6 NTRK3 ETV6-NTRK3.E5N15.COSF571.2 328 357
BTBD1 NTRK3 BTBD1-NTRK3.B4N14 217 433
EML4-ALK.E6aA20.AB374361.1 0 0
CD74-ROS1.C6R34.COSF1200.1 0 0
KIF5B-RET.K24R11.COSF1262.1 0 0
Table 3. Read counts, SeraseqTM FFPE NTRK Fusion RNA Reference Material, 10% dilution into FFPE sample
Grey rows indicate negative controls: fusion isoforms not present in the control sample*Sequencing experiments are performed in two independent occasions.
For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. SERASEQ is a trademark of SeraCare Life Sciences, Inc.
Amplicons are designed strategically targeted for fusion junction.
The assay is designed for use with the Ion Torrent sequencing platforms with comprehensive end-to-end NGS research workflow.
Gene BGene A
5’ 3’ 5’ 3’
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