Why do Archer® FusionPlex® assays use RNA instead of DNA as input material? It all comes down to biological relevance, cost and turn-around time. Translocations can occur anywhere in the genome, including introns and other non-coding sequences. They can also occur within the coding regions of genes with limited expression patterns. What this means is that many of the translocations that occur in a cell may not be expressed and thus have little or no biological relevance. For this reason, DNA is not the ideal substrate to search for oncogenic fusions. RNA, on the other hand, is the intermediate product of gene expression and is ideal for detecting fusions, because you are only looking at those that are expressed and potentially oncogenic. Searching for translocations in non-coding regions of the genome is time consuming and expensive. For example, DNA-based hybrid capture techniques tile over intronic regions, which can be repetitive, homopolymer-prone and span 100kb or more. This approach requires more probes, more space on your sequencer and more input material. And even then, coverage can be spotty. On the other hand, FusionPlex assays use RNA transcripts and place gene-specific primers near known fusion breakpoints, so you can identify translocations with a single primer. And because FusionPlex assays combine primers for multiple fusion targets, you can efficiently detect more fusions with less reads and input material. FusionPlex assays use RNA to detect fusions and are better, faster and cheaper than DNA-based hybrid capture techniques. Detect fusions the Archer way, with one of the many FusionPlex assays.
The level of multiplexing depends on the number of targets and the number of reads generated by the instrument per run. This will vary for each catalog panel as well as custom panels. Custom fusion detection assays will need to be optimized to balance the number of reads needed against the level of multiplexing.
Archer Illumina® or Ion Torrent™ FusionPlex® Panels
|Archer Illumina or Ion Torrent Panel||Input Material||Applications||# of Genes||# of Targets/Assay||Recommended # of Reads|
|Archer FusionPlex ALK, RET, ROS1 Panel v2||RNA/TNA||Fusions/SNVs||3||29||1,000,000|
|Archer FusionPlex Heme v2 Panel||RNA/TNA||Fusions||87||607||1,500,000|
|Archer FusionPlex NTRK Panel||RNA/TNA||Fusions||3||25||1,000,000|
|Archer FusionPlex Sarcoma Panel||RNA/TNA||Fusions||26||148||1,500,000|
|Archer FusionPlex Solid Tumor Panel||RNA/TNA||Fusions/SNVs||53||290||3,000,000|
|Archer FusionPlex Lung Thyroid Panel||RNA/TNA||Fusions||8||42||1,500,000|
|Archer FusionPlex CTL Panel||RNA/TNA||Fusions/SNVs/Expression||35||195||1,500,000|
|Archer FusionPlex Oncology Research Panel||RNA/TNA||Fusions||74||393||3,000,000|
|Archer FusionPlex ALL Panel||RNA/TNA||Fusions||81||506||1,500,000|
|Archer FusionPlex Myeloid Panel||RNA/TNA||Fusions||84||507||1,500,000|
|Archer FusionPlex Pan-Heme Panel||RNA/TNA||Fusions||199||1054||4,500,000|
The expected cluster density will vary by instrument type and panel version. Please see table below for details.
|Instrument||panel||Expected Cluster Density|
|HiSeq®||High Output, TruSeq v3||750-850 k/mm2|
|High Output, TruSeq v4||950-1050 k/mm2|
|Rapid, v2||850-1000 k/mm2|
|MiniSeq||Mid & High Output||170-220 k/mm2|
|NextSeq®||Mid & High Output, v2||170-220 k/mm2|
Please refer to your panel-specific protocol for complete library denaturation and loading instructions.
The expected average size for amplicons will range between 150 and 400 base pairs as viewed on a Bioanalyzer trace. However, you should assume an average fragment length of 200 base pairs when using the KAPA Biosystems® Library Quantification panel for qPCR. Our recommended dilutions and MiSeq® and PGM® input amounts are all based on an assumed average fragment length of 200 base pairs. Please refer to the panel specific protocols for guidance.
The expected average size for amplicons will range between 150 and 400 base pairs as viewed on a Bioanalyzer™ trace. However, assume an average fragment length of 250 base pairs when using the KAPA Biosystems Library Quantification panel for qPCR. Our panel-specific recommended dilutions for the MiSeq and PGM input amounts are all based on an assumed average fragment length of 250 base pairs. Please refer to the panel specific protocols for guidance.
For all extraction methods below we recommend the following elution buffer and methods for quantification and QC:
|Recommended Elution Buffer||Quantification Method||Recommended Quality Check Step|
|nuclease-free water||– Qubit® RNA HS assay panel (Life Technologies® Q32852) for FusionPlex|
– Qubit HS dsDNA (Life Technologies Q32851) for VariantPlex
|– Archer PreSeq® RNA QC Assay (AK0043-16) for FusionPlex|
– Archer PreSeq DNA QC Assay (AK0067-16) for VariantPlex
Total Nucleic Acid Extraction from Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue
|Recommended Extraction Method||Extraction panel Protocol Recommendations|
|Agencourt® FormaPure® Total Nucleic Acid Extraction (A33342)||– DO NOT treat with DNase.|
– Use heat blocks versus water baths.
– Perform a one-hour digestion at 55°C at Step 5. Do not digest overnight.
– Perform reverse-crosslinking by incubating one hour at 80°C between Steps 5 and 6.
– Elute the sample in 40µL nuclease-free water at Step 23.
|Promega ReliaPrep™ FFPE Total RNA Miniprep System (Z1001)||– Extend the reverse crosslinking step at 80°C to one hour.|
|Maxwell RSC RNA FFPE panel (AS1440)||– Do not DNase treat. Proceed directly from 15 minutes at room temperature to centrifugation at full speed for 2 minutes.|
Fresh-Frozen Tissue (FF), Cell Lines and Blood
|Recommended Extraction Method|
|Any total RNA (for FusionPlex) or DNA (for VariantPlex) extraction panel|
Tumor specimens are commonly preserved as FFPE samples. Unfortunately formalin fixation can often cause base deamination, resulting in sequencing artifacts. For example, a cytosine on the negative strand is deaminated into a uracil. In traditional opposing primer-based enrichment, the uracil is transcribed into an adenine and the artifact is amplified during PCR. Because amplification occurs before any type of adapters are added to the amplicons, strand specificity is lost, and therefore the sequence analysis will cause a false-positive C to T single nucleotide variant. On the other hand, Anchored Multiplex PCR-based enrichment identifies these deamination events because Molecular Barcode Adapters are ligated to the DNA prior to amplification. Combined with strand- specific primers, AMP™ maintains the ability to differentiate between positive and negative strand readouts during sequence analysis. So the same C to T transition detected on all negative strands clearly indicates a false-positive SNV, and thus no mutation is called. Let’s take a look at actual sequencing data. If this were data from opposing primer-based enrichment, the prevalence of a C to T transition in an FFPE screen would indicate an NRAS G13D variant prevalent in non-small cell lung cancer. But because AMP preserves strand specificity, all of the C to T transitions were detected on the negative strand, demonstrating with extremely high statistical confidence that this was, in fact, an FFPE deamination. Anchored Multiplex PCR is better than traditional opposing primers because strand-specific priming allows you to identify and correct for deamination events that would otherwise lead to false-positive results.
Using the Qubit® instrument is not recommended for the final library concentration. We cannot guarantee consistent loading concentrations with the Qubit because a size selection of the final library is not performed. Therefore, we recommend using the appropriate KAPA Biosystems Library Quantification panel for accurate quantification of sequenceable molecules. Other commercially available qPCR-based library quantification panels can also be utilized.
Mutations that drive oncogenesis and disease progression come in all forms, including gene fusions, which can be identified and characterized by sequencing a fusion transcript.
Traditional opposing primer based library preparation methods require target and fusion specific primers that define the region to be sequenced. After amplification, adapters are then ligated to the DNA for further amplification and sequencing. The problem with detecting fusions this way is that you need primers that flank the target region and the fusion partner, so only known fusions can be detected.
Anchored Multiplex PCR enables you to detect the target of interest, plus any known and unknown fusion partners. This is because AMP uses target-specific uni-directional primers, along with reverse primers, that hybridize to the sequencing adapter that is ligated to each fragment prior to amplification. With this approach, your target region, plus any known or novel fusion partners, are selectively amplified for sequencing. This increases the analytical sensitivity of your fusion assay by eliminating false negatives due to novel variants or fusions.
MBC Adapters for Illumina®
|Catalog #||Molecular Barcode Adapter (MBC) Description|
|SA0040||Archer MBC Adapters A1-A8 for Illumina|
|SA0041||Archer MBC Adapters A9-A16 for Illumina|
|SA0042||Archer MBC Adapters A17-A24 for Illumina|
|SA0043||Archer MBC Adapters A25-A32 for Illumina|
|SA0044||Archer MBC Adapters A33-A40 for Illumina|
|SA0045||Archer MBC Adapters A41-A48 for Illumina|
|AK0016-48||Archer MBC Adapters Set B for Illumina|
|AK0017-48||Archer MBC Adapters Set C for Illumina|
Barcode Adapters for Ion Torrent™ Platform
|Catalog #||Molecular Barcode Adapter (MBC) Description|
|SA0363||Archer Barcode Adapters 1-8 for Ion Torrent|
|SA0364||Archer Barcode Adapters 9-16 for Ion Torrent|
|SA0365||Archer Barcode Adapters 17-24 for Ion Torrent|
|SA0366||Archer Barcode Adapters 25-32 for Ion Torrent|
|SA0367||Archer Barcode Adapters 33-40 for Ion Torrent|
|SA0368||Archer Barcode Adapters 41-48 for Ion Torrent|