FAQ

AMP™ Chemistry & Workflow

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MBC Adapters for Illumina®
Catalog #Molecular Barcode Adapter (MBC) Description
SA0040MBC Adapters A1-A8 for Illumina®
SA0041MBC Adapters A9-A16 for Illumina®
SA0042MBC Adapters A17-A24 for Illumina®
SA0043MBC Adapters A25-A32 for Illumina®
SA0044MBC Adapters A33-A40 for Illumina®
SA0045MBC Adapters A41-A48 for Illumina®
AK0016-48MBC Adapters Set B for Illumina®
AK0016-48MBC Adapters Set C for Illumina®
Barcode Adapters for Ion Torrent Platform
Catalog #Molecular Barcode Adapter (MBC) Description
SA0363Barcode Adapters 1-8 for Ion Torrent™
SA0364Barcode Adapters 9-16 for Ion Torrent™
SA0365Barcode Adapters 17-24 for Ion Torrent™
SA0366Barcode Adapters 25-32 for Ion Torrent™
SA0367Barcode Adapters 33-40 for Ion Torrent™
SA0368Barcode Adapters 41-48 for Ion Torrent™

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.

Invitae Illumina® or Ion Torrent™ FusionPlex® Panels
Illumina or Ion Torrent PanelInput MaterialApplications# of Genes# of Targets/AssayRecommended # of Reads
FusionPlex ALK, RET, ROS1 Panel v2RNA/TNAFusions/SNVs3291,000,000
FusionPlex Heme v2 PanelRNA/TNAFusions876071,500,000
FusionPlex NTRK PanelRNA/TNAFusions3251,000,000
FusionPlex Sarcoma PanelRNA/TNAFusions261481,500,000
FusionPlex Solid Tumor PanelRNA/TNAFusions/SNVs532903,000,000
FusionPlex Lung Thyroid PanelRNA/TNAFusions8421,500,000
FusionPlex CTL PanelRNA/TNAFusions351951,500,000
FusionPlex Oncology Research PanelRNA/TNAFusions/SNVs/Expression743933,000,000
FusionPlex ALL PanelRNA/TNAFusions815061,500,000
FusionPlex Myeloid PanelRNA/TNAFusions845071,500,000
FusionPlex Pan-Heme PanelRNA/TNAFusions19910544,500,000
FusionPlex Lymphoma PanelRNA/TNAFusions1257162,000,000
Illumina® VariantPlex® Panels
Illumina Panel# of Targets/AssayRecommended # of Reads
VariantPlex Solid Tumor Panel6602,000,000-3,000,000
VariantPlex p53 Panel23100,000
VariantPlex CTL Panel2901,000,000
VariantPlex Core AML Panel105750,000
VariantPlex BRCA1/2 Panel23500,000

Library complexity refers to the input material used for library prep.

  • High complex library: Usually a result of using high quality input material and will only have a few copies for each molecule in the input material. As a result, there will only be a few copies of each molecule after PCR2 (lots of molecules need to be amplified), and thus a low deduplication ratio. Deeper sequencing will help to increase the deduplication ratio.
  • Low complex library: Usually a result of using low quality input material and will only have a few molecules in the input material. As a result, those few molecules will be highly amplified after PCR and lots of copies of each molecule will be present in the final library, and thus a high deduplication ratio.

In order to amplify a biomarker with a traditional primer-based approach (target specific forward and reverse), there is a need to design for both primer binding sites. This means it’s only possible to interrogate fusion that are known about at the time of assay development. Therefore, all rare, new, or completely novel fusions will be missed.

Anchored Multiplex PCR (AMP), on the other hand, amplifies from a one gene specific primer and one universal primer site in the adapter region, which is ligated to all fragments in the starting material. This enables all possible fusions, regardless of the partner, to be amplified during library preparation and sequenced.

We use the library concentration as a metric to determine whether the library passes or fails. The cutoff is 4nM.

  • We do not perform a size selection during our library prep, therefore we capture all fragments, long, medium and short. This will result in a distribution instead of a pronounced peak on the Bioanalyzer™.
  • Sequenceable amplicons can only be quantified by KAPA qPCR as KAPA amplifies off the Illumina® P5 and P7 adapters. As a result, only fragments where both adapters are present will be quantified. This is important, as these are the only fragments that will anneal to the flow cell and can be sequenced.
  • Qubit™ and Bioanalyzer™ will result in too much variation as all DNA fragments will be measured, including the fragments that lack one or both adapters.

In conclusion, quantifying the library pool by KAPA qPCR will achieve better loading results with any Illumina® instrument.

We strongly recommend to a repeat of the KAPA protocol followed by normalizing the library using the results. This ensures that the sequencer is loaded correct and reduces issues with over- or under-loading the sequencer.

We do not recommend using Qubit™ or Bioanalyzer™ to verify the normalized concentration. The Qubit™ often overestimates the concentration, as it measures all DNA fragments, including fragments without P5 and P7 adaptors. The Bioanalzyer™ is an inaccurate method to determine the library concentration and it will raise question regarding the length of the fragments.

You are welcome to use an internal control, but we don’t routinely use a control internally.

Yes, you can use the Thermo Fisher Library Quantification Kit. This kit uses the the P5 an P7 for preparing the amplicons for quantification.

Here is a link to the Collibri™ Library Quantification Kit from Thermo Fisher.

As the KAPA® Library Quantification Kit has been discontinued, we are now recommending the following kit from Thermo Fisher. This kit uses the P1 and A for quantifying the amplicons.

Ion Library TaqMan™ Quantification Kit.

Yes, our libraries can be sequenced on the iSeq; however, one can’t run the Immunoverse™ BCR panel on the iSeq, as the sequencer does not have 2x300bp capabilities. It is possible to run the Immunoverse TCR panel, as this only requires 2x150bp. The maximum number of reads from the iSeq 100 is 4-6M (max 8M) reads, however, we do recommend a 20% PhiX spike-in.

Regarding the loading protocol: iSeq needs about 5x times the loading as on a MiSeq.

It is not possible to pool Invitae libraries with Ion Torrent libraries, as our indices are the same as those of Ion Torrent. Non-Invitae libraries could only be run as long as the indexes do not overlap.

Functional testing and iterative optimization of primer design meets predetermined thresholds for various metrics including primer and coverage uniformity.

The input types and amounts used during testing are representative of the assay’s intended molecule type and workflow.

For FusionPlex designs, in addition to RNA input, DNA input is used to assess primer functionality in the absence of target expression variability.

For de novo primer design, we use conservative metrics to ensure performance in the context of AMP chemistry and high primer specificity. The Assay Designer algorithm does not allow for any primer designs that would bind at another place in the genome other than to amplify the intended target according to a BLAST alignment. Additionally, we consider the 10, 12, 14, and 16bp k-mer count as an indicator of potential off-targeting.

For panels developed using Designer Pro, wet lab generated data is also used to guide primer performance optimization.

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