Archer Frequently Asked Questions

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Archer FusionPlex and VariantPlex FAQs

FusionPlex and VariantPlex General FAQs


Accompanying video available at

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.

For a comprehensive and interactive list of peer-reviewed publications, please visit

For a comprehensive and interactive list of posters presented at scientific conferences, please visit


Accompanying video available at

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

There are no control genes added to the VariantPlex panels.

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.

The control genes for the assays are CHMP2A, RABA7A, GPI and VCP. These are housekeeping genes required for the maintenance of basic cellular function.

Extraction Recommendation FAQs

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 kit (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 Kit 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 Kit (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 kit

Do not use buffers with EDTA. Use nuclease-free water or 10mM Tris-HCl pH 8 for sample storage.

DNAse treatment can reduce the quality of the RNA in the sample. When using FFPE samples, total nucleic acid is the recommended assay starting material.

Input nucleic acid quality and quantity FAQs

The minimum recommended input for Archer VariantPlex assays is best determined by running the Archer PreSeq DNA QC Assay. Depending on the delta Cq score the minimum recommended input will be 50-200ng. Please refer to the following Instructions for Use for more details.

AK0037-8 Archer Universal DNA Reagent Kit v2 for Illumina®
AK0067-16 Archer PreSeq DNA QC Assay

If higher library complexity is desired, the assay can tolerate up to 400ng of DNA.

  • Determine the sample concentration on the Qubit® instrument using the dsDNA HS Assay Kit (Life Technologies® Q32851)
  • Perform the PreSeq™ DNA QC qPCR assay to assess DNA quality and help predict whether or not there is sufficient good quality DNA to proceed with library preparation.

  • Determine the sample concentration on the Qubit® instrument using the HS RNA Assay Kit (Life Technologies® Q32852)
  • Visualize the size distribution and RNA integrity on an Agilent Bioanalyzer RNA 6000 Nano Kit (5067-1511)
  • Universal RNA Kits version 2 (AK0040-8, AK0042-8) come with PreSeq™ qPCR QC to assess RNA quality and help predict whether or not there is sufficient good quality RNA to proceed with library preparation. PreSeq can also be purchased separately from the RNA assay if you would like to screen all RNA samples prior to proceeding through library preparation.

The assay requires RNA fragments to be a minimum of 60 to 70 bases in length. The Universal RNA Reagent kit v2 (AK0040-8, AK0042-8) contains RNA QC primers that will amplify a 113-bp amplicon of the VCP gene. After first-strand cDNA synthesis, the sample can be tested by qPCR to determine whether to continue with the assay. Please refer to the Instructions for Use for more information.

AK0040-8 Archer Universal RNA Reagent Kit v2 for Illumina-8
AK0042-8 Archer Universal RNA Reagent Kit v2 for Ion Torrent-8

Genomic DNA cannot be used with the RNA Universal protocol. Archer Universal RNA Reagent kits are designed to use total nucleic acid or RNA as the starting material. Genomic DNA can be used as starting material for the Archer Universal DNA Reagent kits.

Repeat the nucleic acid isolation to increase quantity of starting material available for the assay.

If higher library complexity is desired, the assay can tolerate up to 250ng of RNA. Too much input will decrease the final library yield.

The minimum recommended input for the assay is 20ng of total nucleic acid. The amount of input material can be adjusted according to the quality of the RNA sample. When using less than 10ng of input material, the number of PCR cycles for the First and Second PCR may need to be increased. Note that reduced sample input may adversely affect read diversity. Please refer to the kit protocol for further instructions and information.

PreSeq FAQs

The Archer PreSeq DNA QC Assay is a qPCR-based assay to evaluate the quantity of amplifiable DNA in a sample in relation to a known Assay Standard. The assay amplifies a 100bp genomic DNA sequence in the sample, as well as the standard, in separate reactions. A comparison of the two resultant quantification cycle (Cq) values results in a DNA QC score. The DNA QC score holds predictive value for library yield, and can be used to gauge the amount of input material required for Archer VariantPlex Assays. Please refer to the following instructions or your kit specific protocol for more information.

AK0067-16 Archer PreSeq QC Assays

The Archer PreSeq RNA QC assay contains primers that target a 113-bp region of VCP, one of the housekeeping control genes targeted in the Archer FusionPlex assays. The 113-bp region spans an exon-exon junction, so background DNA will not interfere with RNA quality assessment. A Cq cutoff is experimentally determined for all RNA samples above which samples will not pass QC on Archer Analysis. Please refer to the following instructions or your kit specific protocol for more information.

AK0043-16 Archer PreSeq QC Assays

Recommended Commercially Available Control FAQs

Yes, we recommend the following as a copy number variation normal control:

  • CNV Normal control – DNA from LCL, CEPH/UTAH PEDIGREE 1463 "Genome in a Bottle" (Coriell Institute, NA12878)

Yes, we recommend the following:

  • Negative control – DNA from LCL, CEPH/UTAH PEDIGREE 1463 "Genome in a Bottle" (Coriell Institute, NA12878)

The "Genome in a bottle" control is also recommended for use as a normal for assays looking for copy number variation.

Yes, there are commercially available controls. We recommend the following positive and negative controls:

FusionPlex Assay Recommended Controls

Panel Fusion Positive Control Fusion Negative Control
FusionPlex ALK, RET, ROS1 V2 ROS1-positive RNA sample, such as the HCC-78 cell line from Creative Bioarray© (CSC-C0569) Ambion® Human Lung Total RNA (Life Technologies AM7968)
FusionPlex Heme V1 High BCR-ABL p210 control that is part of the ipsogen® BCR-ABL Mbcr Control Kit (QIAGEN 670191) Human Blood Leukocyte Total RNA (BioChain R1234148-10)
FusionPlex FGFR V1 Negative BCR-ABL p210 & p190 that is part of the ipsogen BCR-ABL Mbcr Control Kit (QIAGEN 670191) is positive for the FGFR1-FGFR1OP2 fusion. Human Adult Normal Tissue: Colon (BioChain R1234090-50)
FusionPlex Sarcoma V1 ROS1-positive RNA sample, such as the HCC-78 cell line from Creative Bioarray© (CSC-C0569) Human Adult Normal Tissue: Skeletal Muscle (BioChain R1234171-50)
FusionPlex NTRK V1 No fusion positive controls are available to recommend at this time. Human Adult Normal Tissue: Thyroid (BioChain R1234265-50)
FusionPlex Solid Tumor V1 ROS1-positive RNA sample, such as the HCC-78 cell line from Creative Bioarray© (CSC-C0569) Ambion® Human Lung Total RNA (Life Technologies AM7968)
FusionPlex Lung Thyroid V1 ROS1-positive RNA sample, such as the HCC-78 cell line from Creative Bioarray© (CSC-C0569) Ambion® Human Lung Total RNA (Life Technologies AM7968)
FusionPlex Oncology Research V1 ROS1-positive RNA sample, such as the HCC-78 cell line from Creative Bioarray© (CSC-C0569) Ambion® Human Lung Total RNA (Life Technologies AM7968)

Kit Storage and General Protocol FAQs

We recommend that Enhancers (A+B) be added to the GSPs for some of our panels with more difficult targets. Our current stability studies are ongoing. At this point we recommend that the GSP plus Enhancer mix be stored for no longer than 4 weeks.

Yes, you can use the kit if it has been left out overnight at room temperature. However, the pouches must have remained sealed with the desiccant packs inside. Please note that exposure to temperatures above 37ºC may affect VariantPlex kit performance.

Remove all beads if possible. However, a very minimal amount of AMPure beads has no noticeable impact on the assay.

FusionPlex Assays

There are four stopping points in the workflow:

  1. After Second Strand cDNA Synthesis
  2. After Adapter Ligation
  3. After First PCR
  4. After Second PCR

VariantPlex Assays

There are three stopping points in the workflow:

  1. After Adapter Ligation
  2. After First PCR
  3. After Second PCR

FusionPlex Assays

The FusionPlex library preparation protocol takes approximately 9.5 hours of total time, depending on the version of the kit used, and 2.5 hours of hands-on time to process 8 samples.

VariantPlex Assays

The library preparation protocol takes approximately a day to process 8 samples.

Store all components of the Archer Universal Reagent kits between 2°C to 8°C. Archer VariantPlex and FusionPlex assays containing the liquid gene-specific primers (GSP1s and GSP2s) should be stored between -30°C to -10°C.

Storage at 4°C is ideal, however accidental storage of the kit at –20°C will not adversely affect kit performance. Bring to room temperature prior to use.

Library Preparation FAQs

Library Quantification FAQs

The source of the nucleic acid, the purification method, the chemical the nucleic acid is suspended in and the starting amount of nucleic acid can contribute to low library concentration. Recommendations for improving library yield include:

  • Extract TNA from FFPE tissue with one of our recommended methods.
  • Make sure input quantity was measured using the appropriate Qubit® assay.
  • Perform the Archer PreSeq QC assay to determine quality.

Incorrect PCR cycling can also lead to low library yields. Ensure that the cycling temperatures, cycle numbers and ramp rates are accurate. Check the instrument to make sure it is calibrated and in good working order.

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 Kit for accurate quantification of sequenceable molecules. Other commercially available qPCR-based library quantification kits can also be utilized.

Although a template negative control may produce yield, it will fail the QC filter in Archer Analysis and thus will not produce meaningful results.

FusionPlex Assays

Final FusionPlex library concentrations should range between 15nM and 500nM for the v2 Kits. Sequencing libraries with less than 4nM yields is not recommended.

VariantPlex Assays

Final VariantPlex library concentration should range between 4nM and 200nM for the v2 Kits . Sequencing libraries with less than 4nM yields is not recommended.

FusionPlex Assays

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.

VariantPlex Assays

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.

Sample Multiplexing FAQs

The new Archer Ion MBC adapters are identical to the Ion Xpress barcode adapters. It is possible to multiplex together, but take caution not to multiplex identical barcode adapters in the same run.

All Archer MBC adapters are compatible for simlutaneous use on a sequencing run.

Catalog # Molecular Barcode Adapter (MBC) Description
SA0040Archer MBC Adapters A1-A8 for Illumina
SA0041Archer MBC Adapters A9-A16 for Illumina
SA0042Archer MBC Adapters A17-A 24 for Illumina
SA0043Archer MBC Adapters A25-A32 for Illumina
SA0044Archer MBC Adapters A33-A40 for Illumina
SA0045Archer MBC Adapters A41-A48 for Illumina
AK0016-48Archer MBC Adapters Set B for Illumina
AK0017-48Archer MBC Adapters Set C for Illumina

MBC Adapters for Illumina

Catalog # Molecular Barcode Adapter (MBC) Description
SA0040Archer® MBC Adapters A1-A8 for Illumina®
SA0041Archer® MBC Adapters A9-A16 for Illumina®
SA0042Archer® MBC Adapters A17-A24 for Illumina®
SA0043Archer® MBC Adapters A25-A32 for Illumina®
SA0044Archer® MBC Adapters A33-A40 for Illumina®
SA0045Archer® MBC Adapters A41-A48 for Illumina®
AK0016-48Archer® MBC Adapters Set B for Illumina®
AK0017-48Archer MBC Adapters Set C for Illumina®

Barcode Adapters for Ion Torrent Platform

Catalog # Molecular Barcode Adapter (MBC) Description
SA0363Archer® Barcode Adapters 1-8 for Ion Torrent™
SA0364Archer® Barcode Adapters 9-16 for Ion Torrent™
SA0365Archer® Barcode Adapters 17-24 for Ion Torrent™
SA0366Archer® Barcode Adapters 25-32 for Ion Torrent™
SA0367Archer® Barcode Adapters 33-40 for Ion Torrent™
SA0368Archer® Barcode Adapters 41-48 for Ion Torrent™

The Illumina MiSeq will work best when index diversity within a run is high. For example, if eight samples are included in a run, and the user chooses to use only one MBC Adapter paired with eight different MiSeq Index 1 Primers, the run may fail due to low barcode diversity. In this example it is best to use eight different Archer MBC Adapters paired with eight different MiSeq Index 1 Primers. Please refer to the panel specific protocols for guidance.

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 v2RNA/TNAFusions/SNVs3291,000,000
Archer FusionPlex Heme v2 PanelRNA/TNAFusions876071,500,000
Archer FusionPlex NTRK PanelRNA/TNAFusions3251,000,000
Archer FusionPlex Sarcoma PanelRNA/TNAFusions261481,500,000
Archer FusionPlex Solid Tumor PanelRNA/TNAFusions/SNVs532903,000,000
Archer FusionPlex Lung Thyroid PanelRNA/TNAFusions8421,500,000
Archer FusionPlex CTL PanelRNA/TNAFusions/SNVs/Expression351951,500,000
Archer FusionPlex Oncology Research PanelRNA/TNAFusions743933,000,000
Archer FusionPlex ALL PanelRNA/TNAFusions815061,500,000
Archer FusionPlex Myeloid PanelRNA/TNAFusions845071,500,000
Archer FusionPlex Pan-Heme PanelRNA/TNAFusions19910544,500,000
Archer FusionPlex Lymphoma PanelRNA/TNAFusions1257162,000,000

Archer® Illumina® VariantPlex Panels

Archer Illumina Panel # of Targets/Assay Recommended # of Reads
VariantPlex Solid Tumor Panel6602,000,000-3,000,00
VariantPlex p53 Panel23100,000
VariantPlex CTL Panel2901,000,000
VariantPlex Core AML Panel105750,000
VariantPlex BRCA1/2 Panel23500,000

Archer Library Sequencing FAQs

The read depth requirements listed differ between the website and the product insert because the read depth will depend on the desired sensitivity of the end user. The website gives the recommended read depth to capture >95% of all possible SNV transitions with an allele fraction (AF) of 0.05, which is the most common AF desired. The read depth can be modified depending on your preferred sensitivity.

As shown in the left graph below, ~97% of all possible SNV transitions can be called at 0.05 AF when sequenced to a read depth of 1.5M with the VariantPlex® Core Myeloid kit, and thus 1.5M reads is listed on the website for VariantPlex Core Myeloid. If the read depth is doubled to 3M, ~97% of all possible SNV transitions can be called at half the allele fraction, 0.025 AF. For this reason, 3M reads are recommended in the product insert, as some users are interested in detecting sub-0.05 AF SNV transitions with this panel.

A similar trend is shown in the right graph below for the VariantPlex Myeloid kit. A read depth of 3M is recommended on the website as this will enable detection of >95% of SNV transitions at 0.05 AF. However, increasing the read depth to 4M reads will allow detection of >95% of sub-0.05 AF SNV transitions, so this is recommended in the product insert.

Sensitivity vs read depth for Core Myeloid
Sensitivity vs read depth for Myeloid

Low sequence diversity is the source of the erratic signal intensity in the beginning of Read 1 (shown in the first 25bp in the figure below). This low diversity occurs during sequencing of the adapter present in Archer libraries. The MBC adapter has a number of bases that are common for all of your samples. It is that “common” region that contributes to the low read diversity in the first 25 bases. Addition of PhiX to the library pool increases sequence diversity and reduces error rates when sequencing through this region.

Erratic signals shown in the first 25bp of an Illumina sequencing run on an Archer library
Libraries should be diluted to 25pM before loading onto the Ion Chef. As with any library prep/NGS based assay, this value may vary by instrument and library concentration. The suggested range for S5 is 35-60pm and 13-25pM for PGM.

Yes, Archer Illumina Libraries (RNA and DNA) and non-Archer libraries can be multiplexed on the same sequencing run provided the non-Archer libraries are compatible with sequencing under the following conditions:

  1. Nextera®-based sequencing chemistry.
  2. 2x8bp index reads.
  3. 2x150bp paired-end reads

If you have any quesitons about library compatibility please contact technical support at

NextSeq demultiplexing should be done using Illumina's bcl2fastq2 Conversion Software.

Observe the following:

  • Be sure to use the "--no-lane-splitting" option mentioned on page 28 of the Illumina's bcl2fastq2 Conversion Software v2.19 Guide. The default output from the software splits the data by lane for each sample, resulting in four sets of fastq files (one for each lane) per sample. A single set of fastq files is required for each sample uploaded to Archer Analysis. Therefore, using the "--no-lane-splitting" option is critical.
  • Note that Archer Analysis has the ability to automatically process demultiplexed sequencer data through the Watched Folders mechanism. Since every sequencing environment is different, you will need to provide a small linker script which copies your paired fastq files to the location(s) specified for the Watched Folder(s). Your Archer representative can help you with more details.
  • The reverse complement of the P5 adapter sequences provided by Archer are needed to successfully demultiplex the NextSeq run.

Download Archer adapter sequences and their reverse complements file here.

Yes, Archer RNA and Archer DNA libraries can be multiplexed on the same sequencing run. Note, however, that the bp size adjustment applied to the quantification calculation for DNA libraries is 250bp, while for RNA it is 200bp.

All Archer libraries require Nextera® based sequencing chemistry and use 2x8bp index reads, and 2x150bp paired-end reads.

Yes, Archer Illumina libraries are compatible with NextSeq and HiSeq as long as they are run on a paired-end flowcell using Nextera® chemistry. Please note that our libraries are dual indexed and should be sequenced using 2x150bp PE reads and 2x8bp index reads. Please refer to the panel specific protocols for guidance.

The recommendation for how much PhiX to add to your VariantPlex library pool will vary depending on the panel used. Please refer to the Instructions for Use for your VariantPlex Panel for the recommended percentage of PhiX.

AK0051-8 Archer VariantPlex Solid Tumor Panel
AK0069-8 Archer VariantPlex p53 Panel
AK0071-8 Archer VariantPlex CTL Panel

The recommended amount of PhiX will depend on the instrument type and kit version. Please see table below for details.

Instrument Kit Version Recommended amount of PhiX to include on the run
MiSeq® V2 5%
V3 15%
NextSeq® V2 20%

Refer to the kit specific protocol for complete library denaturation and loading instructions.

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 750-850 k/mm2
High Output, TruSeq v4 950-1050 k/mm2
Rapid, v2 850-1000 k/mm2
MiSeq® v2 1000-1200 k/mm2
v3 1200-1400 k/mm2
MiniSeq® Mid & High Output 170-220 k/mm2
NextSeq® Mid & High Output, v2 170-220 k/mm2

Please refer to your kit-specific protocol for complete library denaturation and loading instructions.

Refer to the table below for fragment size estimates for different applications.


Application Recommended starting fragment length (bp)
FusionPlex® 200
Immunoverse™ 250


Application Recommended starting fragment length (bp)
VariantPlex™ 250
Reveal ctDNA™ 150

Software FAQs

Archer Analysis

Archer Analysis: General

There are three ways you can access Archer Analysis:

  1. You can use our live demo site at: However, this is meant for training and demonstration, and is not for analysis of confidential samples. It is also used by other customers and staff and may become very backed up.
  2. The recommended method is to download the Archer Analysis virtual machine. You will need to install virtualization software (VMware) and download our .ovf file, which can be imported by the virtualization software.
  3. You can purchase Analysis Unlimited, which is a private cloud-based instance of Archer Analysis where you pay only for the data you use. Contact technical support at for more details.

Contact for further information and to request the user guide.

Yes, we do offer a standalone version of Archer Analysis for purchase.

Analysis Unlimited is a highly scalable, private cloud-based instance of Archer Analysis. With this solution, your fees are based on your use.

For more information contact

The web-based demo version of Archer Analysis is not explicitly secure, though it does benefit from many of the security features of amazon web services. All other forms of Archer Analysis are designed to be secure for use within an organization.

Analysis Unlimited operates in a virtual private cloud, which is accessible only through the dedicated IP addresses you provide. ArcherDX will not access your data without your permission.

Your downloaded virtual machine (VM) is a local installation and operates within your institution's existing security infrastructure.

There are three different options for accessing Archer Analysis:

  1. You can download the software free of charge and install it locally on your own machine. Contact to download Archer Analysis.
  2. You can purchase secure cloud-based access from ArcherDX. For more information please contact
  3. You can purchase a server that comes pre-loaded with Archer Analysis. For more information please contact

Visit for complete information regarding Archer Analysis.

Yes, this is one of the benefits of using virtual machines. You can have a virtual machine with each version of the software enabling version lock-down and ease of validation.

However, there is no way to migrate data from one version to the other. So, if you are keeping older data after updating to a new version of the software you will need to keep the older version in order to access it, or you will need to re-process the FASTQ files with the new version of the software.

While most current browsers are supported, we recommend using Chrome, FireFox, or Safari. Note that older versions of Internet Explorer do not support multiple selections of FASTQ files in the file dialog box; however, most recent versions of Internet Explorer work correctly. Because we continue to find incompatibilities with some versions of Internet Explorer, we do not recommend this browser at this time.

The software can be set up to run samples either in parallel or in serial with the maximum number of concurrent samples depending on the amount of memory allocated to the software on your local machine. For Archer Analysis Unlimited, the maximum number of concurrent samples can be set when your virtual private cloud is deployed or at any time after deployment by ArcherDx.

In general, we require 1 CPU and 12GB of RAM per concurrent sample.

For example if you allocate 1 CPU and 12GB of RAM to Archer Analysis, you will be able to process one sample at a time. If you allocate 2 CPUs and 24GB, this would allow you to process 2 samples simultaneous, and so on.

The downloadable virtual machine is configured to run 1 sample at a time by default. This is easily adjusted: Within the VM you must edit the /root/nodes file and change the np=1 value to np=X, where X is the number of samples you wish to process concurrently. Shut down the VM after editing this file and adjust your VMWare settings to match the formula above (1 CPU and 12GB RAM per concurrent sample). You must of course have enough real hardware on the host to support the desired configuration. Restart the VM and it should now process the number of concurrent samples you specified.

Archer Analysis: Software Installation

Please contact technical support at to request a link to download the Archer Analysis Virtual Machine Installation Guide and/or User Guide. Additionally, informational videos and links to other supporting materials can be found at

If you already have the virtual machine or a login to our demo site, you can find the documentation under the help menu at the top of the screen.

For installations requiring only a single sample to be processed at a time, we have extensively tested Oracle VirtualBox (for workstations). Oracle VirtualBox can be downloaded from However, because VirtualBox does not properly support multiple processors, we require VMWare on any installation where more than 1 sample needs to be processed at a time.

For single workstation solutions requiring multiple sample processing, VMware Workstation Pro is recommended for Windows, and VMware Fusion is recommended for Mac. However these VMWare products support a maximum of 64GB RAM to be allocated.

For installations requiring more than 5 samples to be run concurrently, we recommend VMware EXSi.

VMware products can be downloaded from

Users must have a virtualization application, such as Virtual Box for single sample processing, or VMware for multiple concurrent sample processing, installed on their system first. The system must have at least 12GB of free RAM (beyond whatever else the host itself is using) and enough disk space to install the virtual image and upload/process some number of samples (we recommend at least 200GB to start). The virtual image contains all of the analysis functionality in a single package and does not require any additional software other than the previously mentioned virtualization host.

This functionality is enabled through a mechanism called "Watched Folders". A user with the appropriate permissions may configure a directory on the server for Archer Analysis to monitor. When sample files or a folder of sample files appear in the watched directory, Analysis will automatically process these files according to the Watched Folder template.

Typically a demultiplexer would be responsible for performing read assignment and copying sample files to these watched folder locations. This is beyond the scope of Archer Analysis itself but we can provide assistance in helping you configure this in some scenarios.

Please see the user manual for instructions on setting up this feature. For a copy of the Archer Analysis User Manual contact

Archer Analysis: Getting Started

A targeted mutation file is a text file format (extension: .vcf) containing a list of variants of interest, primarily single nucleotide variants (SNVs), short insertions and short deletions. Variants are listed in the file by chromosomal position, reference and alternate sequences which define each variant. Users supply Archer Analysis with a targeted mutation file before initiating an analysis job; Archer Analysis version 4.0 utilizes this information to further annotate variant results and optionally narrow the focus of variant calling for both the RNA SNP/InDel and DNA SNP/InDel analysis pipelines.

  1. Open your virtualization software.
  2. Hit the start button on the virtual machine you wish to run. A console will appear as the virtual machine starts up and will provide an IP address.
  3. Type the IP address from your console into your browser to run Archer Analysis.

This depends entirely on how your local network infrastructure is configured and where you have installed Archer Analysis. Archer Analysis is configured to request an IP address via DHCP, thus the address it receives is controlled by your own network environment.

In most cases, your computer and the Virtual Machine will obtain a non-routable IP address. This means that you are only accessing resources within and behind your firewall or proxy.

For advanced installations, the Virtual Machine can be configured to conform to other addressing scenarios. Your network administrator can help reconfigure this if necessary.

The analysis of a single set of FASTQ files (~250,000 reads, or 50-100MB data) typically takes less than an hour. Larger samples can vary considerably, but typically take between 1.5-3 hours, depending on the library complexity, number of reads, and analysis options chosen. Some samples of around 3 million reads can take upwards of 7-8 hours; this is largely driven by significantly greater than normal library complexity.

By default, the VM runs each sample serially, but the system can be set up to process multiple samples at the same time. Consult the Archer Analysis User Guide for instructions on setting up Archer Analysis for parallel processing of samples.

Contact for further information and to request the user guide.

This will depend on your network speed and is not limited by Archer Analysis itself. Consult with your local IT department for more information.

This will depend on your internet speed and file size. You can check your upload speed at, and calculate your upload time by dividing the sum of your file size by your upload speed. You will also see a progress bar as your samples are uploaded.

Use to calculate your internet speed to determine how long it will take to upload your files

In this example, the speed is 44.38 megabytes/second. You would therefore divide your file size by this number to determine how many seconds it would take to upload to Archer Analysis.

We recommend that you put all of the FASTQ files to be uploaded into a single location. Note that all of the files for a single job must be uploaded at the same time. Make sure to select and upload both the read 1 and the read 2 FASTQ files for each sample when working with the Illumina® paired-end reads libraries. Do not allow your machine to go to sleep during file upload.

See how to start a new job and upload FASTQ files to Archer Analysis in the instructional video below:

Accompanying video available at


In Archer Analysis you have the ability to re-run or clone a job. These differ in the following ways:

Job re-runs always run under the original job owner's account. This will delete the original analysis results and reprocess using either the original job settings or the job owner's current settings (you will be asked which you would like to use when re-running the job).

Cloning creates a copy of the analysis and leaves the original data and job intact. This option always clones the job into the current user's account (e.g. if user Bob ran an analysis but user Sally clones Bob's job, the cloned job will belong to Sally and run using either Bob's original settings or Sally's current settings). Additionally, cloning a job allows other parameters to be changed with respect to the analysis type and even which samples within a job are cloned.

Archer Analysis: Expected Metrics


How can I view and filter variants in Archer Analysis?

Variant reporting is even more powerful in Analysis 4.0. You can now apply specific variant filters, customize how and what information is displayed, and save filter sets for repeated use.

From the sample summary screen, click “Detailed summary” below the sample name. Then, click on “Variant Summary”. From here you can control your filter sets, add filters, hide or display columns, or export your data.

First, create a new filter set by clicking the dropdown and selecting “new”. We’ll call this one “somatic with targets”. After this filter set is saved, you can start adding some filters. For certain filters, such as this consequence filter, you can select multiple values by holding down the command key on a mac or the control key on windows while clicking. You can create as many filters as you want, and when you’re done, hit save.

Next, click “edit columns” to change the visibility and sorting of each column. You can also view information about that specific field, and when you’re done, simply click “save”.

Archer Analysis now makes it easier to view VEP and VCP annotations within variants. Simply hover over the Annotation icon, and Analysis displays the related annotations. You can even sort based on whether annotations are present or not, and set the default annotation for each variant call.

After you’ve organized your data the way you want, you can choose to export only your filtered data in TSV format or download the source file containing all of your variants in VCF or TSV format. This way, you can view your variants in the external application of your choice.

With Archer Analysis, it’s never been easier to organize variants and take control of your data. Learn more about Archer Analysis and test-drive it today at

Unless you have updated your analysis settings, your sample will not show “PASS” in the QC section. The minimum number of unique reads is set to ensure there are no false negatives. Any fusions detected with a failed QC call may be real if they are classified as strong fusions; but fusion negative samples with a failed QC call may be too degraded or have too few reads to detect the fusion. Each lab can determine experimentally during validation whether or not this threshold needs to be adjusted.

This has proven to be a reasonable criterion for the success of the assay. However, users are encouraged to evaluate the results themselves and set their own cutoff.


Archer Analysis utilizes BWA and Bowtie 2 for mapping to hg19. Any read that has an Alignment Score less than 30 is removed from consideration. Reads with a mapping quality less than 30 are either multiple mapping reads or contain many low-quality bases (low FASTQ base quality). For the exact calculation of the mapping quality and the factors involved, please refer to the BWA and Bowtie 2 documentation. Note that this default value can be adjusted by the user under Analysis Settings in Archer Analysis.



  • A DNA read is a read that spans an intron/exon junction.
  • An RNA read is a read that spans an exon/exon junction.
  • An ambiguous read is a read that does not span any junction and therefore cannot be identified as DNA or RNA.

On Target percent is the percentage of library fragments that include the entire GSP2, which requires that the fragment be long enough to reach the GSP2 and be of high enough quality at the end of that read to identify all bases in the GSP2.

This reduces the total number of fragments that are analyzed and may cause the sample to fail QC. Poor quality samples have lower percentages of unique fragments. Make sure you are following the extraction recommendations.

The score to pass alignment is 30 by default, but may be changed in the settings menu. This score is essentially the Smith-Waterman score from BWA-MEM, which, for example, gives a +1 for each perfect match and a –1 for each mismatch in the read. For example, with the default score, there may be 30 perfectly matched bases, or 32 matches with 2 indels, or 33 matches and 3 indels, etc.

If a fusion is in the Archer Quiver known fusion database, we always display it in the Strong Evidence tab.

In all other de-novo cases, the read event must meet the following criteria:

  1. Have a minimum of 5 unique (de-duplicated), breakpoint-spanning reads that support the gene fusion.
  2. Must not be flagged as a likely run cross-contamination event.
  3. At least 10% of the total reads for that GSP2 must support the fusion over the wild-type transcript.
  4. Have at least 3 unique start sites; breakpoint-spanning reads that support the gene fusion.
  5. Be an exon-exon fusion. Intron-exon fusions will be displayed in the weak evidence bin by default.
  6. Fusion partners cannot have homologous sequences. Fusion partners with too high sequence similarity will be flagged as likely mispriming events.
  7. Fusion partner genes must not be known paralogs.
  8. Fusion must pass additional alignment quality criteria (BLAST scores, mismatches in alignments, number of fusion partners involved, number of bases aligned to each fusion partner) to appear in the Strong Evidence bin.

These are the default values, however users can adjust these values in the Analysis User Settings based on their own data.

For FusionPlex RNA Assays: A sample will pass the QC filter if the average number of unique RNA start sites per control GSP2 (Gene Specific Primer 2) is 10. This metric is reported under the Read Statistics tab, labeled Average Unique Start Sites per GSP2 Control.

For VariantPlex DNA Assays: A sample will pass the QC filter if the average number of unique DNA start sites per target GSP2 is 50 or above. This metric is reported under the Read Statistics tab, labeled Average Unique Start Sites per GSP2.

This has proven to be a reasonable criterion for the success of the assay. However, users are encouraged to evaluate the results themselves and set their own cutoff.

This percentage varies with library quality. For high quality libraries, 80-99% of the total fragments should pass the adapter trimming filter.

A molecular barcode is a unique 8-bp sequence that is part of the MBC Adapter. and is used to identify unique fragments and “de-duplicate” the sequencing reads from a sample. This, along with the random start sites, helps identify and remove PCR duplicates.

Molecular barcodes are an important element of Archer Anchored Multiplex PCR (AMP) chemistry. Watch the video below to learn more about how molecular barcodes work.


Accompanying video available at

Archer Analysis uses symbols on the summary and evidence pages to indicate known fusions, show QC metrics, denote fusion partner sequence similarity and show if a fusion is an exon/intron fusion

Means the fusion passed all QC metrics

Means the fusion is a known fusion according to the Quiver database

Means the fusion and the exact breakpoint are known according to the Quiver database

Means the percent fusion reads for the GSP2 detecting that fusion is below the set threshold

Means the fusion partners show sequence similarity to each other and could therefore be a mispriming event

Means the fusion is an exon/intron fusion. Most fusions are expected to be exon/exon fusions.

Means the unique start sites for this fusion was below the threshold of MIN_UNIQUE_START_SITES_FOR_VALID_FUSION (default=3).

Represents a transcriptional read-through event. The fusion event is the results of a gene transcribing into the next gene downstream of it.

Means there is expression imbalance within the gene.

Means there is likely intra-run sample contamination that induced this fusion call.

These genes are known Ensembl paralogs

This annotation met at least one of the following criteria

  1. Too many mutations between reference and consensus
  2. The maximum expect score returned by BLAST across all segments is too high
  3. More than two genes were found in the consensus
  4. Insufficient number of bases aligning to each fusion partner

Each symbol has a tool-tip associated with it. Hover your mouse over any of these in the Archer Analysis UI for additional information.

We have packaged a job summary report (accessible from the reporting icon at the job summary level) and a sample summary report (accessible from the reporting icon at the sample summary level) with Archer Analysis. In addition, you can create and install your own reports for further customization.

Archer Analysis: Troubleshooting

Archer Analysis 4.0 now employs a de novo assembling strategy for all fusion and oncogenic isoform calling. While this can produce results that vary in statistics from prior versions, it's proven to be more accurate and should result in fewer ambiguous calls. Since this is a parameterized option, it can be toggled on or off in user settings (we recommend on for the reasons listed above).

Some benefits of the de novo assembling strategy are:

  1. We see more consistency in read clustering. Because de novo alignment depends only on the similarity of reads to each other and not to the genome (in the first step), the alignments surrounding the breakpoint tend to be longer and of higher quality.
  2. Annotation is more robust because the de novo alignment reduces the occurrence of mis-aligned and semi-repetitive reads to an incorrect genomic location.
  3. De novo alignment usually leads to more "collapsed" fusion calls. That is, in version 3.3 and version 4.0 you can sometimes get the same fusion breakpoint reported multiple times due to slight differences elsewhere in the reads, but away from the actual breakpoint location. The issue is less prevalent in version 4.0, although it may still occur if a fusion is called by more than one GSP2. This means that you don't have a given number of supporting reads split out over multiple calls of the same fusion, thus the metrics for the collapsed fusion have more reads, more unique start sites and a higher percent GSP2. Ultimately, this leads to higher confidence calls.

Archer has provided information to guide the detection of fusions and variations. However, every sample is different. Each lab must interpret the results, and take into consideration any information known about their particular samples. Read statistics and the visualization tools can assist in making proper calls. Some things to consider include:

  1. Does the fusion appear in the strong evidence tab?
  2. Are there several reads supporting the fusion call? 5 is the MINIMUM recommended number of unique reads to call a fusion.
  3. Are there enough reads supporting the fusion transcript(s) as compared to the wild-type transcript(s)?
  4. Is the expression level of the target gene normally high or low?
  5. How does the expression level of the target in the analyzed sample compare to expression in a normal (control) sample?
  6. Is the fusion known in the literature?
  7. When visualizing the data, are there any insertions or deletions present in more than a few of the reads near the breakpoint? This may indicate a mapping error.

Watch the video below to learn more about visualizing reads and fusions in JBrowse:

Accompanying video available at


Run QC is most affected by starting library quality, library complexity, and coverage. You will see the effects of these factors on “Average Unique Start Sites per GSP2” for VariantPlex assays, and "Average Unique Start Sites per GSP2 Control" for FusionPlex assays. This is found under the DNA/RNA Statistics section of the Read Statistics tab in Archer Analysis. The default setting is 50 Average Unique Start Sites per GSP2 for VariantPlex assays, and 10 Average Unique Start Sites per GSP2 Control for FusionPlex assays. These default values are adjustable, and you may want to change the cutoff based on your validation results.

DNA/RNA statistics in Archer Analysis

Click on "Detailed Summary" to get more information about why it failed and decide if you want to accept the data, change parameters, or re-run the sample.

Read about what is acceptable data in these other FAQs:

  1. What is the most common reason runs fail QC?
  2. How do I know if my fusion is real?

Make sure that you have:

  1. Selected the correct assay type (DNA, RNA, or QC)
  2. Selected the correct sequencing platform option (Illumina® paired-end is the default)
  3. Selected the correct target region (Gene Target File)

If both of these are correct, then contact Archer technical support at or 877-771-1093.

Consult the Analysis Logs by selecting the “View Analysis Logs” option (Archer Analysis Logs icon ) to view the log files to obtain clues about why the error was produced. If possible, send the log files to ArcherDX technical support to help in the error analysis.


Currently you cannot, but we are working on adding that function. For now, if you find a novel fusion, contact tech support at

Quiver provides information on which of our panels include a particular gene or fusion, as well as which exons participate in the known fusion, and links to PubMed and other public resources. This is useful for researching fusions that you are unfamiliar with or when designing your own custom assay.

The Quiver Fusion Database is a compilation of known fusions from several public databases. Visit to learn more about it.

Assay Designer

No. ArcherDx’s Anchored Multiplex PCR (AMP) chemistry enables you to detect any fusion partner to your target of interest. You do, however, need to specify the exon of interest for each target as well as the direction of the fusion partner from the target exon (3’ or 5’). Note that mid-exon breakpoint primer design is not supported by Assay Designer at this time. If you want to detect mid-exon breakpoint fusions, contact technical support at

Assay Designer assumes that all breakpoints are at the specified end of the exon (3’ or 5’). If you want to detect mid-exon breakpoint fusions, contact technical support at

After the design has been approved and submitted for order, you can expect to receive your equimolar primer pools in 6-8 weeks.

There is no specific maximum number of targets for Assay Designer. However, we will flag panel designs that we do not believe in silco design is sufficient to meet your expectations or ours. In this instance we will be in contact with you to discuss specifics and how you would best like to proceed with procuring a custom panel.

To provide you with the best chance of creating a successful primer design for your assay, a member of our team will carefully review each primer in your design and remove any primers expected to perform poorly. In some cases, we might recommend that the custom panel be redesigned by our in-house R&D team to ensure that they meet both your and our expectations.

There is no current minimum. However, the standard 8 controls included in our catalog panels will be included in any design.

No. Primers are designed in silico and ordered directly through Assay Designer. They are not tested in any way prior to shipping. They will arrive in an equimolar pool. For best results, it is recommended that you test the primer pool on DNA before use for fusion detection. Testing the primers on DNA will allow you to confirm that all primers are fully functional without the added complication of expression levels associated with target amplification of RNA. If you are interested in having ArcherDx functionally test your primers and balance your primer pools, contact technical support at

No. The primer search setting in Assay Designer are fixed so that primers are optimal and likely to be fully functional with our assay. If you need assistance recovering important targets, contact technical support for assistance at

Assay Designer might fail to design a primer to a target region for one or more of the following reasons:

  1. Target region has high similarity to other regions in the genome, which would result in off-targeting
  2. GC content in target region is too high or too low
  3. There are known mutations present in the primer site

A custom fusion panel can be made at Exons from genes can be selected and added to the custom fusion panel. It is also possible to upload a .csv file with the appropriate information. Once the design has been submitted, a representative from ArcherDX will contact you to make sure the design is correct. The Quiver Fusion database ( can also be used to find known fusions that can then be added to the custom design.

Assay designer is our free online tool used to create your own custom assays. It can be found here:

For further assistance, please contact Archer Technical Support


Phone: +1 (877)-771-1093 (toll-free) or +1 303-357-9001

Limitations of Use

For Research Use Only. Not for use in diagnostic procedures.

This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.

© 2016 ArcherDX, Inc. All rights reserved. Archer™ and Archer™ FusionPlex™ are trademarks of ArcherDX, Inc. Illumina® HiSeq™, NextSeq™, MiSeq®, and Nextera™ are registered trademarks of Illumina, Inc. Agencourt®, AMPure® and FormaPure® are registered trademarks of Agencourt Biosciences Corporation, a Beckman Coulter company. Life Technologies™, Ambion®, Ion Torrent™, SYBR®, Applied Biosystems®, GeneAmp®, Veriti® and DynaMag™ are trademarks of Thermo Fisher Scientific, Inc. Qubit® is a registered trademark of Molecular Probes, Inc. KAPA Biosystems® is a registered trademark of KAPA Biosystems, Inc. Bio-Rad®, T100™, C1000†, S1000† and iTaq™ are trademarks or registered trademarks of Bio-Rad Laboratories, Inc. Bioanalyzer™ is a registered trademark of Agilent Technologies, Inc. RNase Away® is a registered trademark of Molecular Bio-Products, Inc. ipsogen® and QIAGEN® are registered trademarks of QIAGEN GmbH.

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