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Frequently asked questions (FAQs)

Whatever it takes to get information to you we are willing to go that extra mile. Whether it’s product related queries, guidance on lab procedures, or technical questions, you can find them here.

You can also contact us at techsupport@devyser.com for more information. 

General

What is the difference between handbook and Instructions for use (IFU)? Which one should I use?

Handbooks are the protocols for the Research Use Only (RUO) version of our kits. These documents can be freely downloaded from our website in the respective product page (scroll down until the ‘Downloads’ section and select ‘Guidelines and handbooks’ to find the download link). Instructions for use (IFU) are the protocols for the certified version of our kits (under European CE-IVD or IVDR certifications). To download an IFU, you will need an access code that is printed on the box label, like in the figure below. In the ‘Downloads’ section under ‘Instructions for use’, you can enter the access code and click ‘Get Files’ to download the IFU.

 

Can I aliquot the activated mix?

As stated in the IFUs, we advise against aliquoting the activated mix, because this can lead to suboptimal results as tested by our R&D.

What are the validated sample types for Devyser kits?

The table below provides an overview of the sample types validated by our R&D team for each of our kits (other types might still compatible, but the user should do their own validation). For the most complete, please see the respective handbook/IFU of each kit under ‘Sample Requirements’. In case of doubt, please contact techsupport@devyser.com.

 

Devyser Kit

Category

Validated sample type

Compact v3

FA (QF-PCR)

Human gDNA extracted from whole blood, amniotic fluid and chorionic villus biopsy samples.

Complete v2

FA (QF-PCR)

Human gDNA extracted from whole blood, amniotic fluid and chorionic villus biopsy samples.

Extend v2

FA (QF-PCR)

Human gDNA extracted from whole blood, amniotic fluid and chorionic villus biopsy samples.

Resolution

FA (QF-PCR)

Human gDNA extracted from whole blood, amniotic fluid and chorionic villus biopsy samples.

AZF v2, RUO, Extension

FA (Allele-specific)

Human gDNA extracted from whole blood

CFTR Core, Italia

FA (Allele-specific)

Human gDNA extracted from whole blood

CFTR68

FA (Allele-specific)

Human gDNA extracted from whole blood

HFE v2

FA (Allele-specific)

Human gDNA extracted from whole blood

Thrombophilia

FA (Allele-specific)

Human gDNA extracted from whole blood

CVD

FA (Allele-specific)

Human gDNA extracted from whole blood

BRCA

NGS

Human gDNA extracted from whole blood/FFPE samples

HBOC

NGS

Human gDNA extracted from whole blood

Thalassemia

NGS

Human gDNA extracted from whole blood

CFTR

NGS

Human gDNA extracted from whole blood/Blood spots

FHv2

NGS

Human gDNA extracted from whole blood

Chimerism

NGS

Human gDNA extracted from whole blood

RhD

RT-PCR

cfDNA extracted from maternal plasma

Which are the recommended DNA extraction kits for Devyser products?

Please see the table below for several extraction kits that we have been tested and validated for our different kits. 

 

Extraction kit 

NGS Kits (excluding Chimerism and Accept cfDNA) 

QF-PCR Kits 

Allele-specific Kits 

RT-PCR Kits (RHD) 

QIAamp DNA Blood Mini Kit 

All 

CFTR Core, AZF (RUO), CVD, HFE, Thrombophilia 

 

QIAsymphony DSP DNA Midi Kit 

All, except HBOC  

 

AZF v2/Extension, CFTR 68, 

 

QIAamp DNA FFPE Tissue Kit 

BRCA 

 

 

 

High Pure FFPET DNA Isolation Kit 

BRCA 

 

 

 

QIAamp DNA Mini Kit 

CFTR Core for dried blood spots, HBOC 

Chorionic Villus Samples 

 

 

QIAamp DNA 

Blood Mini QIAcube Kit 

 

 

AZF v2/Extension, CFTR 68 

 

QIAsymphony DSP Virus/Pathogen Midi Kit 

 

 

 

MagNA Pure 96 

 

 

 

Under evaluation 

 

Which PCR instruments are recommended?

Please see in the table below the PCR instruments that we have tested and validated for our different kits.

 

PCR Instrument

NGS Kits

QF-PCR (all kits)

FA Kits

RT-PCR Kits (RHD)

ABI GeneAmp System 9700

Chimerism

X

CFTR Core, Thrombophilia

 

Thermo Fisher Scientific Veriti

X

 

AZF v2/Extension, CFTR 68, CVD, HFE, Thrombophilia

 

Thermo Fisher Scientific VeritiPro*

X

X

X

 

Applied Biosystems 7500 Real-Time PCR System

 

 

 

X

QuantStudio 6/7 Flex Real-Time PCR System

 

 

 

X

Roche LightCycler 480 II Real-Time PCR System

 

 

 

X

*Please download the VeritiPro addendum under the respective product page.

NGS

How many samples can I run on the different NGS instruments/flow cell?

Please consult our coverage calculators, which can be found on our website.

Can I combine libraries generated with different Devyser kits?

Pooling libraries different Devyser kits must be validated by users according to their standards and processes. Please contact techsupport@devyser.com if you have questions.

 

How should I pool libraries of germline and somatic samples generated with the Devyser BRCA kit?

Libraries generated from germline samples can be pooled prior to purification. In contrast, libraries from somatic samples should be purified separately before being pooled. For more information on how to pool and clean up somatic samples, please see our Technical Note here.

If you want to combine germline and somatic libraries, please use our Devyser Sequence Coverage Calculator to obtain the volumes to spike in from both the somatic and germline normalized library pools.

Which sequencing modes are validated for Devyser kits?

This information can be found in the respective kit IFU or handbook under ´Sequence Data Analysis´ or ‘Sequencing Mode’.
An overview for all Devyser NGS kits can be found below.

 

Kit 

Sample type 

Sequencing Mode

Devyser BRCA 

Germline, Somatic 

SR 1x301, PE 2x151 and PE 2x251 

Devyser BRCA PALB2*

Germline, Somatic

PE 2x151

Devyser HBOC 

Germline 

PE 2x151 

Devyser Thalassemia 

Germline 

PE 2x151 

Devyser CFTR 

Germline 

SR 1x301, PE 2x151 

Devyser FH v2 

Germline 

PE 2x151 

Devyser LynchFAP*

Germline 

PE 2x151

*BRCA PALB2 and LynchFAP are Research Use Only kits. The information is verified, not validated.

 

Which Illumina Sequencers have been evaluated by Devyser?

An overview for all Devyser NGS kits can be found below. For assistance on platforms that are not listed, please contact techsupport@devyser.com.

 

Illumina Sequencer

NGS Kits

Illumina MiSeq

BRCA, BRCA PALB2*, HBOC, Thalassemia, CFTR, FHv2 and LynchFAP*

Illumina MiniSeq

CFTR, Thalassemia, BRCA

Illumina iSeq 100

CFTR, BRCA

*BRCA PALB2 and LynchFAP are Research Use Only kits. For all other assays the use of indicated Sequencers has been validated (CE-IVD).

What is the total number of samples that can be processed with the Devyser Library Clean kit?

The Devyser Library Clean kit is used in the purification step of our Devyser NGS products. The number of library pool purifications that can be performed can vary depending on the NGS kit as listed in the table below. Please note that a library pool can contain up to 96 PCR2 libraries (when using Index Plate A).

 

Kit

Number of purifications

Devyser BRCA

12

Devyser BRCA PALB2

12

Devyser CFTR

12

Devyser FH v2

6

Devyser HBOC

16

Devyser LynchFAP

12

Devyser Thalassemia

6

What is the average library size for Devyser NGS kits?

Please see the table below for the theoretical average library sizes for each kit. These values are based on the average between the lengths of the amplicons generated during PCR 1 and 2.

 

Kit

Average length (bp) amplicon PCR1

Average length (bp) amplicon PCR2

Devyser BRCA

230

340

Devyser BRCA PALB2

228

338

Devyser CFTR

222

332

Devyser FHv2

267

377

Devyser THAL

234

344

Devyser HBOC

218

328

Devyser LynchFAP

243

353

 

 

What is the recommended concentration for the various Devyser libraries (prior to the execution of the Illumina denaturation protocol), and what is the equivalent concentration in nM?

The recommended concentration for the purified library pool in ng/µl is indicated in the respective IFU or handbook for the kit. The table below shows the corresponding concentration in nM, using the following equation, utilizing the theoretical average amplicon size (PCR2) of the assay.

 

 

 

MiSeq

MiniSeq

iSeq

Kit

ng/µl

nM

ng/µl

nM

ng/µl

nM

Devyser BRCA

0.33-0.41

1,47-1,83

-

-

-

-

Devyser BRCA PALB2

0.15-0.22

0.67-0.99

-

-

-

-

Devyser CFTR

0.33-0.41

1,51-1,87

0.22

1,00

0.22

1,00

Devyser FH v2

0.25-0.30

1,00-1,21

-

-

-

-

Devyser THAL

0.25-0.30

1,10-1,32

0.23

1,01

-

-

Devyser HBOC

0.23-0.27

1,06-1,25

-

-

-

-

Devyser LynchFAP

0.20-0.25

0.86-1.07

-

-

-

-

 

What is the recommended minimal coverage for the assay?

In general, the recommended minimal coverage per amplicon is 100x for SNV and 200x for CNV for all Devyser NGS assays (except FH v2, Chimerism, and Accept cfDNA). For all assays, the respective IFU or handbook converts this to a minimal coverage per sample, as per the Devyser Sequence Coverage Calculator. This online tool also assists in planning the sequencing run, namely the the number of samples that fit on a given Illumina sequencing instrument and flowcell.

Why did my Illumina sequencing run over or under-cluster?

Clustering issues with Illumina runs are generally due to incorrect loading concentration of sequencing libraries. However, other reasons could include:

  • Inaccurate library quantification – quantification should be done using a Qubit instrument and assay kit as described in the respective IFU or handbook. Fresh standards should also be prepared and measured for each preparation, and reagents must be at room temperature. Other methods such as NanoDrop and Tapestation/Bioanalyzer are not as precise as fluorometric approaches (e.g., Qubit), so we strongly advise against using them for library quantification.
  • Inefficient library denaturation – always prepare freshly diluted NaOH for denaturing libraries prior to sequencing. The NaOH stock solution must have a pH >12.5 prior to any dilution.

It is important to consider that under-clustering is normally preferable to over-clustering, as it affects data quantity than quality (except on MiniSeq). If in doubt, or when first optimizing the assay, please aim for the lower end of the recommended loading concentration as stated in our IFUs to avoid over-clustering.

How many analyses can be performed when one kit is purchased?

The same number of analyses can be performed as tests purchased (i.e., number of reactions in a kit or kits). For example, if an 8-test kit is purchased, you can perform 8 analyses with our software.

My analysis status on Amplicon Suite is stuck in uploading/analysis?

Please ensure that you have a stable internet connection and that the FASTQ files are stored locally. Make sure the software is installed locally and not on a server. If the issue persists after one re-upload attempt, please contact techsupport@devyser.com.

How can I open an account for Amplicon Suite?

To request access to the Amplicon Suite software, please contact your local sales representative. Our team will contact you with installation instructions and account credentials. Please find a list of our distributors here.

Am I using the latest version of Amplicon Suite?

You can check the version you are currently using in the ‘About’ tab in the Amplicon Suite software. If there is an update available, you will normally be prompted to update when starting the software. Also, there is always a notification in the ‘News’ tab when a new version is released. There, you can see the update details, namely the release notes.

Can multiple users login to the same Amplicon Suite account simultaneously?

Yes, multiple users can simultaneously access the same account with the same login credentials.

FA

Do you provide settings for Thermo Fisher’s GeneMapper?

Yes. Devyser Fragment Analysis kits have been validated with GeneMapper versions 4, 5, and 6. The settings files can be found on ‘Software settings’ in the ‘Downloads’ section in the respective product page on our website. For other versions, please contact techsupport@devyser.com.

Why is there an extra peak in a specific Bin?

There could be several causes, for further assistance please refer to our troubleshooting guide and the respective IFU.

How do I avoid crosstalk?

If you experience crosstalk, please reduce the DNA input if the signals seem too high, and if the issue persists, redo spectral calibration and ensure that the calibration passes. For further assistance, please learn more in our troubleshooting guide.