Questions & Answers

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What is the difference between Nextera DNA Flex and Nextera Library prep kits?

Nextera DNA Flex library prep kit provides the flexibility of use across a wide range of genomes from small genomes (microbial genomes, amplicons) to more complex genomes (eukaryotes and human). 

Nextera DNA Flex library prep is rooted in an innovative bead-linked transposome (BLT) complex that tagments genomic DNA by fragmenting and adding adaptor tag sequences in a single reaction step. Once saturated with input DNA, the BLT complex fragments a set number of DNA molecules, providing flexibility to use a wide DNA input range, consistently tight fragment size distribution, and normalized libraries. Following the tagmentation step, a limited-cycle PCR step adds Nextera DNA Flex-specific index adapter sequences to both ends of the DNA fragment, enabling single- and dual-indexed sequencing of pooled libraries across all Illumina sequencing platforms. A subsequent Sample Purification Bead (SPB) cleanup step then prepares libraries for use on an Illumina sequencer.

Is Nextera DNA Flex compatible with Nextera DNA or Nextera XT indexes?

Although they share sequence compatibility, it is not recommended to use XT indexes with Flex libraries as Nextera DNA Flex has indexes of higher purity that have been carefully optimized for improved performance. 

Is Nextera DNA Flex compatible with Nextera DNA or Nextera XT reagents?

No, Nextera DNA Flex reagents are not compatible with Nextera DNA or Nextera XT reagents as the enzymatic chemistry of DNA Flex is very different from that of Nextera DNA and Nextera XT.

As performed with Nextera XT, can we perform bead-based normalization (BBN) with Nextera DNA Flex libraries?

Normalization in Nextera DNA Flex happens during tagmentation, hence there is no need to perform bead-based normalization at the end of library prep. Furthermore, the Nextera XT bead-based normalization process is not compatible for use with Nextera DNA Flex.

What happens if a customer uses Nextera XT’s BBN reagents with Nextera DNA Flex?

This will not produce viable sequencing libraries. The Nextera XT reagents are not compatible for use with the Nextera DNA Flex protocol. 

Can we use Nextera libraries for downstream enrichment capture?

No. Nextera DNA Flex chemistry is incompatible with the enrichment chemistry.


What is included in this kit?

This kit includes all the reagents needed for generating complete libraries ready for sequencing. The kits also include magnetic sample purification beads for sample cleanup steps. For more information, see the Kit Contents section of the Nextera DNA Flex Library Prep Reference Guide.

What consumables and equipment do I need?

A full list of user supplied items can be found in the Consumables and Equipment section of the Nextera DNA Flex Library Prep Reference Guide.

Can other thermal cyclers be used with the Nextera DNA Flex DNA Library Prep protocol?

Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, the Bio-Rad C1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers may differ in their performance which may impact genomic coverage.

Can I use Zymo beads instead of Sample Purification Beads provided in the kit for the cleanup steps?

The kit includes a sufficient volume of Sample Purification Beads reagent to process the intended number of samples for the kit. Do not use beads or columns from any other manufacturer with this protocol.


What blood tubes have been tested?

Prep is optimized for the use of K2-EDTA and K3-EDTA tubes (manufactured by BD). Other blood tubes may work, but they have not been thoroughly validated.

The following additional tubes have also been tested (1 sample for each tube, tested on the same day as collection).

  • Lithium Heparin
  • Sodium Heparin
  • NaF/EDTA
  • NaF/Oxalate
  • Sodium Citrate
  • PAXgene DNA Blood

What is the appropriate temperature for storage and transport of blood tubes?

Store and transport at 2-8°C. Perform lysis reactions within 3 days of blood being collected for optimal results.

Can frozen blood be used?

This has not been thoroughly validated.

Can clotted blood samples be used?


Can I use Nextera DNA Flex kit on blood spots on cards?

Yes, Nextera DNA Flex can be used on blood spots on cards.

How much DNA is there in 10µl of blood?

This is dependent on white cell count of the blood sample. A sample with average white cell count (4-7 x 106 leucocytes per mL blood on average for healthy individuals) the estimated amount of DNA in 10µl of blood is 200-600ng.

What happens if I add more or less than 10ul of blood to the lysis reaction?

This is not recommended as it may affect the efficiency of the lysis reaction which will ultimately impact the amount of DNA obtained and therefore the final library yield. 

Can I use Nextera DNA Flex kit on finger stick? Is there enough blood on one single finger stick? How to prevent blood clot prior to the Flex reaction?

Nextera DNA Flex has not been fully validated with blood from a finger stick.  As a note, Nextera DNA Flex will not work on clotted blood.


Can I use saliva that has been collected in a different collection tube?

This protocol has been developed to work with the Oragene saliva collection tubes. Saliva samples collected into other tube types may not be stable under the same conditions and therefore the use of other collection tubes is not recommended.

Can we use buccal swabs?

The buccal swab sample type has not been tested with this protocol.  Oragene saliva tubes are the only collection method that has been tested.

Following collection, how long are the samples stable for?

Samples are stable at room temperature for up to 5 years. Refer to information from DNA Genotek.

What happens if the saliva tube is under or over filled?

The sample may be less stable due the stabilization reagent in the lid of the tube being at a different final concentration in the sample.

Can I freeze the saliva samples?

Oragene/saliva samples may also be stored at -20°C or -80°C. Samples may undergo at least three freeze-thaw cycles with no evidence of DNA degradation. Refer to information from DNA Genotek.

Does the 50°C incubation of the saliva sample need to be performed immediately prior starting the lysis protocol?

No, the incubation can be performed at any point prior to beginning the prep. Following the 50°C incubation step the sample is stable at room temperature for up to 5 years.

How long does the 50°C incubation need to be?

Samples should be incubated for at least 1 hour but this incubation can also be performed overnight.

What is the purpose of the liquid in the lid of the Oragene Saliva collection tube?

This reagent stabilizes the saliva sample.

How much of the DNA obtained from the saliva sample is human in origin?

The amount of bacterial DNA present is variable between different samples and donors. In approximately 80% of the samples that were tested at least 80% of the DNA obtained aligned to the human reference sequence.

How much DNA is obtained from 30ul of saliva?

The amount of DNA in a saliva sample is very variable between samples and donors. Based on crude Qubit reading from saliva samples, assuming 50% of the DNA is captured during lysis step, the amount of DNA ranges from 34 ng to 2.7 ug. 

What happens if I add more or less than 30 ul of saliva to the lysis reaction?

This is not recommended as it may affect the efficiency of the lysis reaction which will ultimately impact the amount of DNA obtained and therefore the final library yield.

What are the safe stopping points in this protocol when working with blood and saliva?

There are 3 safe stopping points in the protocol:

  • After cleaning up the lysed blood and saliva sample sat 2° to 8°C for up to 3 days
  • After PCR at 2° to 8°C for up to 3 days
  • After PCR Clean Up at -15° to -25°C for up to 30 days


How do I assess the quality of input DNA?

Assess the quality of genomic DNA by running an aliquot of the sample (approximately 10–100 ng) on a 1% agarose gel stained with SYBR Stain. High quality, intact genomic DNA appears as a high molecular weight band (> 10,000 bp) in the absence of a lower molecular weight smear. Low molecular weight smearing can indicate the presence of RNA or degraded DNA. Avoid resuspending DNA In EDTA containing buffers such as TE (similar to Nextera DNA/Nextera XT). 

What method is recommended to quantify my DNA for the Nextera DNA Flex protocol?

For DNA inputs between 100–500 ng, accurate quantification of the initial DNA sample is not required, and normalization of the final yield is expected.

If you are using less than 100 ng DNA input, we recommend quantification.  To quantify input genomic DNA and DNA in the PCR and enriched libraries, use a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. The concentration of gDNA can be determined using the Qubit dsDNA BR Assay or the Qubit dsDNA HS assay. These assays use a fluorescent dye that is highly selective for double-stranded DNA over RNA and can detect samples in a concentration range from 10 pg/μl – 1000 ng/μl. PicoGreen dye can also be used to accurately measure the DNA concentration.

For more information, see the DNA Input Recommendations section of the Nextera DNA Flex Library Prep Reference Guide.

Is the substrate for tagmentation exclusively dsDNA? Will ssDNA/RNA work?

Nextera DNA Flex is optimized for gDNA (dsDNA) and will not work on ssDNA/RNA.

How much input genomic DNA is required for the Nextera DNA workflow?

The Nextera DNA Flex protocol is compatible with DNA inputs ranging from 1–500 ng. For human DNA samples and other large complex genomes, the recommended DNA input is between 100–500ng. For small genomes, the DNA input amount can be reduced to as low as 1 ng (modifying the PCR cycling conditions accordingly).

  • 100ng to 500ng recommended for all large genomes (eg, human)
  • ≥100ng for normalized libraries

What is the minimum amount of DNA to use without DNA quantification (to use the built-in normalization)?

≥100 ng

What happens if < 100 ng DNA input is used for a human sample?

Library yield is not normalized.  Reduced diversity/increased duplicates.

What happens if < 100 ng genomic DNA that is not human is used?

Additional PCR cycles can be implemented, as detailed in the user guide.

What happens if I use more than 500 ng DNA input?

Although testers have successfully generated libraries from more than 500 ng input DNA, that application is not supported.

Does Illumina recommend any DNA extraction method?

Any commercial method that is capable of extracting quality dsDNA is probably effective.

Has Illumina tested GC-rich or AT-rich genomes? Was there any bias?

Yes. A variety of genomes have been tested and the data remains consistent.  A variety of genomes, prepared with Nextera DNA Flex, from bacteria, plants, agriculture, and human can be found on BaseSpace.

Are degraded or FFPE samples compatible with this protocol?

The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is very difficult to reliably predict the quality of a library prepared from FFPE samples using this protocol. Illumina does not support FFPE or degraded DNA as input for this this protocol. This does not mean that FFPE samples cannot be attempted, but that failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.

Can PCR amplicons be used as input?

Yes. The PCR amplicon must be > 150 bp. Shorter amplicons can be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end.


How long does it take to generate ready-to-sequence Nextera DNA Flex libraries?

It takes 2 to 3 hours from genomic DNA input until libraries are ready to load on the flow cell.  The time is dependent on the number of libraries that are prepared.

How many samples can I process at a time?

1 to 96 samples can be processed at a time through the protocol. 

How do I choose index combinations for my libraries if I only pool a small number of samples?

For more information, see the Index Adapters Pooling Guide.  This guide details out the plexing options for different plexity and different sequencers.

Can I use my own set of indexes during PCR?

It is not recommended to use any other indexes as the indexes in the kit have high purity and have been carefully optimized to result in even index representation across libraries.

What is the difference between SPB and SPRIs?

SPB is the acronym for Sample Purification Beads and SPRI is the acronym for Solid Phase Reversible Immobilization.  These two terms are synonymous.  Illumina has transitioned from using the SPRI terminology to SPB.

What is the purpose of double-sided bead purification after PCR?

The purpose of the double-sided bead purification is to size select the library fragments; the two step process first removes the large fragments and the second step removes the small molecular weight fragments.

For more detailed information about Sample Purification Bead Size Selection and Best Practices go to the online training sequencing courses to view the online TruSeq Sample Purification Bead Size Selection and Best Practices.

Why is my sample frothing? How can I stop it frothing? Will frothing ruin my library?

Samples froth because of a detergent used in the library prep. As long as the wash step is included (as described in the protocol), the libraries will not be affected. If desired, gentle pipetting up and down will reduce the amount of froth.

I missed a wash step, is this a problem?

There may be a problem if detergents present in upstream reagents carry over.

Can I use different PCR plates?

Yes, but the protocol has been optimized for the recommended lab ware in the protocol.

Can I use different magnets?

Cleanup procedures have only been optimized and validated using the magnetic stand specified in the Nextera DNA Flex Library Prep Reference Guide. Comparable performance is not guaranteed when using other magnets. Other magnets can be used, but test how long samples need to sit on the magnet as times may vary from the protocol.

Are Flex libraries stranded?

No, because it is a double stranded DNA library and both strands are sequenced.

How can I use Unique Dual Indexes (UDIs) with Flex?

Unique dual indexes from 2 to 96 plex is supported by the 96-index plate configuration. Refer to the Index Adapters Pooling Guide to get details for unique dual indexing of samples. 

IDT for Illumina Nextera DNA UD Indexes uses 10 base pair index codes, whereas Nextera DNA CD Indexes uses 8 base pair index codes. Therefore, you may need to adjust your sequencing setup. For more information, see IDT for Illumina Nextera DNA UD Indexes.

Can I improve indel recall and precision?

Indel precision and recall is impacted by both library prep and the informatics approach used to call variants. In some cases, you may see improved indel recall and precision by reducing PCR cycles, but this will also result in correspondingly lower yield. If too many PCR cycles are removed, there may not be enough library yield for sequencing.

What happens if I am below saturation? Will the library prep still work with similar quality?

Yes, but you will need to quant the library product.

How far above and below the DNA recommended input has been tested?

Libraries have been successfully generated outside of the recommended input range of 1 to 500ng, but is not recommended and therefore done at the customer’s risk.

I left my BLT on the magnet too long. Can I still proceed?

Yes, you can still proceed.

What happens if my BLT beads are dry? Should I still proceed?

No, if the BLT is dry then you should not proceed.

Are the index sequences publicly available?

Yes, they are available in BaseSpace and the Illumina Adapter Sequences Document.

My SPB are not clearing on the magnet. What should I do?

Let them incubate for a longer period until the beads clear.

I over-dried my SPB. What should I do?

Resuspend carefully by pipetting up and down several times, and then proceed with the protocol.

I did not dilute my SPB as directed in the protocol, what should I do?

You will see very low yield. If you kept the beads, you could try to elute DNA and redo cleanup. However, this is not supported.

I want a different insert size; how should I do this? Can I just change the SPB volumes for the second part of the double-sided bead purification?

Yes, you can adjust the usage of SPB but it is not supported.

Can I just do a single sided 1.8x (or more) SPB concentration?

This is not recommended: the assay is less robust and will see much more variable insert size and index representation.  As stated above, customers may adjust the usage of SPB, but doing so is not supported.

My BLT beads look different after 1 wash. What should I do?

Proceed as long as beads are ‘clearing’ after incubation on the magnet.

If I transfer some BLT beads after PCR, will my library be ok?

Visually inspect and re-clear the supernatant on the magnet if you think you transferred some beads.

Can I still quant and normalize my libraries if I use over 100 ng/saturating input?

Yes, but we do not see added benefit in normalization after beads are saturated.

I added too much/too little BLT. What should I do?

This is not recommended or supported. Results are not guaranteed, libraries may still be generated but the yield may be lower and normalization of library yields may not be achieved. Some sequencing metrics such as Diversity may also be reduced’

How many times can I freeze thaw the reagents? (Resuspension Buffer, Tagmentation buffer, Enhanced PCR Mix, and Index adapters)?

We have tested frozen reagents up to 4 freeze/thaws of frozen reagents without observing any loss of activity.

How long can the Nextera DNA Flex tagmentation reaction remain at 10°C hold after the 55°C for 15 mins?

This has not been recommended as a safe stopping point, so it is recommended to proceed with the protocol as recommended.

What factors can cause over- or under-tagmentation with Nextera DNA Flex library prep? Has this been seen in development?

None.  This issue should be completely mitigated with this approach.

After Amplify Tagmentation DNA method, samples were stored at -20°C. The reference guide says store at 2-8°C for up to 3 days. How does this affect my final library?

It is recommended to follow the protocol for safe stopping points and temperature. Storing at -20°C leads to an additional freeze/thaw step.

After Clean-up Libraries method, samples were sealed with Microseal 'B'. What affect does this have on my library and sequencing data? The reference guide says to use Microseal 'F'.

Microseal 'B' is ambient to -40°C and Microseal 'F' is ambient to -70°C, so samples can probably be stored safely.  However, you may have difficulty removing Microseal 'B' after storage, which can cause sample splashing & contamination.  Therefore, Microseal 'F' is recommended for storage.  Also, Microseal 'B' has shown static properties that can cause sample to stay stuck on the seal regardless of centrifugation attempts.

What indexing strategy is used in these libraries?

Nextera DNA Flex Library Prep offers 2 Indexing options.

Index Kit Type Kit Configurations:

  • 20018707 Nextera™ DNA CD Indexes (24 Indexes, 24 Samples)
  • 20018708 Nextera™ DNA CD Indexes (96 Indexes, 96 Samples)

For more information on dual index principles and sample pooling, see the Nextera DNA Flex Library Prep Reference Guide and the Nextera DNA Flex Pooling Guide.

Library Evaluation

What method is recommended to quantify the final library prior to sequencing?

When using DNA inputs of 100-500ng, the normalization features of this library protocol mean that quantification and normalization of individual libraries, generated within a single experiment, is not necessary. There may, however, be slight variations in final yields between different library preparation events, so to achieve optimal cluster density we advise you to pool your libraries with equal volume and quantify the pool.  There can be variations between library preparation events/experiments and users, therefore, when pooling samples of different experiments, it is recommended to quantify & normalize the libraries individually before pooling.

What can cause my Nextera DNA Flex library to look different than the example shown in the Nextera Library Prep Reference Guide?

The profile of the amplified library product can look different from the example shown depending on the type and quality of input DNA. However, the use of BLT and a double-sided size selection helps in maintaining the product length.  However, inaccurate pipetting during the double-SPRI will cause fragment size variation.  Some variation is expected in the profiles of different libraries.

My Nextera DNA Flex library has a bump/shoulder/peak at 1000-3000 bp. Is this ok? What is it?

It is not atypical to see a small bump/shoulder/peak at 1000-3000 bp.  The appearance of the peak can vary between DNA samples and does not affect downstream sequencing. We do not know what this peak is and data is unaffected by this peak. 

How do I check the quality of my library?

Use a Fragment Analyzer (Advanced Analytics) or Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of a tagmented sample. For examples of Bioanalyzer traces and library size distributions, see the Nextera DNA Flex Library Prep Reference Guide. Variation in the Bioanalyzer profiles is expected because it is dependent on the input DNA type.

What is the expected library yield after PCR if the input DNA is over 100ng?

Actual yield may vary based on sample quality and sample type, typically for good quality DNA sample the expected library yield is greater than 4 ng/ul and within an experiment the CV of library yield is <20%.

What is the typical size range and average size of final library?

600bp average fragment length on Bioanalyser (150-1500bp size range).

350bp median insert size from sequencing.

How do I calculate concentration (nM) of my library?

((concentration in ng/μl) / (660g/mol∗average library size)) ×10= concentration in nM


What kit configurations are supported for automation?

Methods are written and tested by our automation partners to support both 24 & 96 sample kit configurations, including a full plate run with up to 96 reactions.  The 96-sample kit is designed to be automation-friendly, including additional volumes necessary for automated processing.

Do the tubed Index configuration of the Nextera DNA Flex kit support automation?

No, automation is only supported by the 24 plex and 96 plex Index plates provided by the Nextera DNA flex kit.

Where can I find the Illumina Qualified methods available for running Nextera DNA Flex?

Illumina Qualified methods are developed, distributed and supported by our Automation Partners. You can learn more about our Partnership program and partners by visiting the web page.

Which automated liquid handlers can be used with the Nextera DNA Flex Library Kit?

All of Illumina’s Automation partners are currently developing methods for one of more of their existing automation platforms.  Please contact the individual partners for more information.  To view a list of our Partners, please visit our Automation Partnership web page.  In general, Nextera DNA Flex is designed to be compatible with most automated liquid-handling systems.

Are the reagent fill volumes suitable for automation?

All reagents provide sufficient overage volume to support automation requirements in the 96-sample kit configuration.  Additionally, the 96-sample kit configuration should support at least two runs of 48 samples each (2 runs x 48 samples).

We recommend utilizing Illumina Qualified* methods from our partners, benefits include:

  • Reduced development & implementation costs.
  • Utilize tested & standardized methods.
  • Choose solutions from leading vendors in laboratory automation.

*Illumina Qualified indicates that libraries prepared with this method have been shown to perform comparably to those prepared manually.


Which Illumina sequencing instruments are compatible with this library prep?

These libraries can be run on all currently available Illumina sequencing systems.  Please see the Nextera DNA Flex Pooling Guide for details.

Should I check for color balance for index reads?

Please see the Index Adapters Pooling Guide to make sure your sequencing set up is appropriate for your sequencing platform. 

What is template loading concentration for various platforms?

  • HiSeq X: 200-300 pM
  • HiSeq 4000: 200-300 pM
  • HiSeq 2000/2500 High Output: 12 pM
  • HiSeq 2500 Rapid Run: 8.5 pM
  • NextSeq: 1.2-1.3 pM
  • MiSeq: 12 pM
  • MiniSeq: 1.2-1.3 pM

How many human libraries could be pooled together to get enough reads for approximately 30x genome coverage?

  • HiSeq X: 8 libraries
  • HiSeq 4000: 6 libraries
  • HiSeq 2000/2500 High Output: 4 libraries
  • HiSeq 2500 Rapid Run: 1 library on 2 flow cells
  • NextSeq: 1 library


Could we pool libraries from 2 independent experiments?

Yes, but the libraries will need to quantified before pooling. If input DNA was at least 100 ng, libraries from each independent experiment may be pooled and quantified together prior to pooling with the second experiment. It is not recommended to pool all samples from independent experiments prior to quantification due to variability observed from user to user.

Are data sets available?

Yes, there are examples of data from HiSeq sequencing runs in BaseSpace.  There are a range of BaseSpace data sets available from microbes, larger eukaryotes (plants, bovine, etc.), and human.

What software versions are required to sequence my prepared libraries?

  • IEM (if needed): Illumina Experiment Manager 1.14 software or later
  • cBot software (if needed): cBot 1.4 software or later
  • HCS software for HiSeq/HiScanSQ: HCS 1.5/RTA 1.13 software or later
  • SCS software for Genome Analyzer: Genome Analyzer SCS 2.10/RTA 1.13 software or later
  • HiSeq Analysis Software (HAS): any version
  • MiSeq Control Software (MCS): 2.2 software or later
  • MiSeq Reporter Software (MSR): 2.2 software or later

Which flow cell can be used with these libraries?

A paired-end flow cell from any Illumina sequencing system can be used with these libraries.

Are the indexes and sequencing primers in the Nextera DNA Flex kit the same as the ones in the Nextera and Nextera XT library prep kits?

IDT for Illumina Nextera DNA UD Indexes, Nextera CD Indexes, and Nextera XT indexes are not compatible with each other.  It is not recommended to interchange these index sets.

Why are the sequencing intensity profiles wobbly in the first ~15 cycles of my run?

This is a normal characteristic of tagmented libraries and relates to the sequence context at the tagmentation site.


How can I add the Nextera DNA Flex library kit to the MiniSeq Local Run Manager analysis software?

Download the Nextera DNA Flex library kit definition file from the Downloads page of the Nextera DNA Flex support site to a known location such as the desktop. Open the Local Run Manager application and select the Gear (System Settings) icon. From here, select Library Prep Kits and select the Add Library Prep Kit option. Navigate to the location of the template file and add the file. The new kit will now appear under the Library Prep Kit dropdown menu when creating a run.  

How can I set Local Run Manager to trim the adapter sequences for this kit during analysis?

During run creation, select the Show advanced module settings option. From here select the Add Custom Setting option. For read 1 adapter trimming, enter “Adapter” as the option name and the appropriate sequence. For Nextera DNA Flex the sequence is CTGTCTCTTATACACATCT.