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 expected cluster density will vary by instrument type and kit version. Please see table below for details.
Instrument
Kit
Expected Cluster Density
HiSeq®
High Output, TruSeq v3
High Output, TruSeq v4
Rapid, v2
750-850 k/mm2
950-1050 k/mm2
850-1000 k/mm2
MiSeq®
v2
v3
1000-1200 k/mm2
1200-1400 k/mm2
MiniSeq®
Mid & High Output
170-220 k/mm2
NextSeq®
Mid & High Output, v2
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 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
Fusions
87
607
1,500,000
Archer FusionPlex NTRK Panel
25
Archer FusionPlex Sarcoma Panel
26
148
Archer FusionPlex Solid Tumor Panel
Fusions/SNV's
53
290
3,000,000
Archer FusionPlex Lung Thyroid Panel
8
42
Archer FusionPlex CTL Panel
Fusions/SNVs/Expression
35
195
Archer FusionPlex Oncology Research Panel
74
393
Archer FusionPlex ALL Panel
81
506
84
507
Archer FusionPlex Pan-Heme Panel
199
1054
4,500,000
Archer FusionPlex Lymphoma Panel
125
716
2,000,000
Archer Illumina Panel
VariantPlex Solid Tumor Panel
660
2,000,000-3,000,00
VariantPlex p53 Panel
23
100,000
VariantPlex CTL Panel
VariantPlex Core AML Panel
105
750,000
VariantPlex BRCA1/2 Panel
500,000
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 Kit 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 Kit 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
Recommended Extraction Method
Extraction Kit Protocol Recommendations
Agencourt® FormaPure®
Any total RNA (for FusionPlex) or DNA (for VariantPlex) extraction kit.
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.
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.