Here we describe a sensitivity experiment we conducted with a custom panel we designed to detect fusions in ALK, NTRK1, 2 and 3, RET, ROS1 and other genes that are often found to be involved in various lung cancers. We were interested to find how sensitive the Archer™ FusionPlex™ Assay was to detect the common SLC34A2-ROS1 fusion product.
RNA was extracted from cell line HCC-78, containing the SLC34A2-ROS1 fusion and was diluted 1:1000, 1:100 and 1:10 in a background of Ambion© Lung RNA to a total RNA content of 200ng. In addition, a library was created of 100% background as the negative control. Libraries were created with the Archer Universal RNA Reagent Kit v2, using the custom target panel containing 119 targets for ALK, NTRK1, 2 and 3, RET, ROS1 and other genes and were sequenced on an Illumina™ NextSeq™. Reads from the 4 lanes were combined, giving an average read depth of about 600,000 read pairs.
To analyze the data in the Archer Analysis software, complimentary software suite provided by ArcherDX, we used the FASTQ files that are created by the demultiplexing software from Illumina™. To ensure that the Archer Analysis software is aware of the location of the 119 targets, we uploaded the FusionPlex-CustomPanel-ARR-NTRK GTF file to the Archer Analysis software using the Manage Custom Target menu item in the Settings menu.
Once the GTF file is uploaded to the Archer Analysis server, we created a new analysis by selecting the PEFORM ANALYSIS button in the main menu. Since this is an RNA Fusion assay, we selected the RNA Fusion option in the RNA Assay Type section and selected the newly created target region
"FusionPlex Custom Panel ARR-NTRK" as the Target Region. This data was run with the Illumina Paired-End library protocol, so we left the default selection for Platform as "
Illumina (paired)" and pressed "
Submit Analysis" to start the analysis.
Figure 3. A new analysis was started using the 8 FASTQ files and the newly created FusionPlex Custom Panel ARR-NTRK target region
After a short time, the run finished successfully and the results page shows a summary of the complete run (Figure 4). From the summary we see that the negative control has no Strong Evidence fusions, while all three of the ROS1 positive dilutions show the SLC34A2-ROS1 fusion. The little bullseye target shows that the breakpoint between the ROS1 and SLC34A2 exons was exactly as it was previously described in the literature and was found in the Archer Quiver database of known fusions.
Figure 4. The job summary page shows the results of the analysis of the 4 ROS1 dilution experiment
We show that it is possible to detect the SLC34A2-ROS1 fusion when it is expressed at the level at which it is expressed in cell line HCC-78 in a background of 1 in a 1000 (0.1%) at the recommended sequence depth. The sensitivity of RNA fusion detection is highly dependent on the expression level of the fusion of interest, the number of targets in the assay and the level of PCR-duplication in the library (which, in turn, is dependent on the quality of the input sample). Low quality (i.e., FFPE samples) often result in low amounts of amplifiable RNA and this experiment shows that even for low amounts of quality RNA, it is possible to detect gene fusions with the Archer Anchored Multiplex PCR kits.
Archer™ Archer™ FusionPlex™ are trademarks of ArcherDX, Inc. Illumina® and NextSeq® are registered trademarks of Illumina, Inc. Ambion® is a registered trademark of Thermo Fisher Scientific, Inc.
Thon has over 15 years of bioinformatics experience. Thon received his Ph.D. in Molecular Biology from the Free University of Amsterdam and worked for Genomic Health, GeneSpring, Avadis NGS and Vector NTI before joining the ArcherDX team.
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