Illumina DNA PCR-Free FAQs

Illumina DNA PCR-Free data can be evaluated with the DRAGEN Germline Pipeline. Other pipelines may be used but may provide divergent results. 

The adapter trimming sequence for both Read1 and Read1 is:

CTGTCTCTTATACACATCT+ATGTGTATAAGAGACA

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.

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

For FFPE, we recommend using a PCR-based kit, such as Nextera DNA Flex. However, for high quality FFPE, Illumina PCR-Free may be suitable.  

Yes.  See the application note titled “Microbial whole-genome sequencing with Illumina DNA PCR-Free Prep, Tagmentation.”

Frozen blood is not supported.

High quality libraries will be generated from 25ng to 2000ng. However, for optimum performance, and when sample amount permits, we recommend using saturating amounts of DNA (at least 300ng for gDNA and recommended raw sample volumes for integrated extraction).

There is elution step from BLT-PF beads after adapter ligation with diluted HP3 (NaOH) which is responsible for libraries become DNA single-stranded.

Rather than modifying the 660g/mol, we use a 2-fold adjustment in the loading concentration to compensate for the difference of quantifying with either Qubit (ssDNA) or qPCR (dsDNA).  The user must be aware of what their method is measuring to understand the correct loading concentration.

The output from this calculation provides the dsDNA molarity equivalent, which makes transition to other instruments and established loading concentrations more directly comparable.

It is compatible only with the 10 bp IDT® for Illumina® - DNA/RNA UD Indexes (formerly the IDT® for Illumina® Nextera DNA Unique Dual Indexes).

Up to 192 UDIs will be supported at product launch. Additional UDIs will be available later in 2020.

No, these are not interchangeable.

IPB adjustments have been done to obtain enough library of as low as 350bp and up to 550bp fragments. For more details, please see the application note titled “Tunable insert sizes with Illumina DNA PCR-Free Prep, Tagmentation.”

Standard input workflow with thermal cycler should be used for 100-299 ng of DNA input. The low input workflow with thermal cycler should be used with 25-99 ng of DNA input. For input between 300-2000 ng of DNA, either the standard input with thermal cycler or standard input with Hybex may be used.

Whole blood, dried blood spots, and saliva should use thermal cycler workflow, and specific instructions for these inputs are marked throughout the Reference Guide. Please see Reference Guide for full details.

At product launch, there will be an Illumina Preset Protocol (IPP) for this library prep kit. 

We recommend storage of individual samples up to 7 days.  Longer storage times may be possible but are not supported by Illumina.

Use of Read 1 custom sequencing primer is required for NovaSeq SBS v1.0 reagents. Failure to use this will cause library sequencing failure. Custom primers (Read 1 or Read 1 + Index 2) are required for all other supported instruments and reagents. The only exception is NovaSeq SBS v1.5 reagents, which already contain the required custom primers. See the bulletin titled “Custom Primer Requirements for the Illumina DNA PCR-Free Prep, Tagmentation Kit” for details.

In regards to handling, it is important to retain the correct fraction (beads or supernatant) where indicated. IPB is highly viscous and certain reagents, such as ST2, TWB, and BLT-PF, are prone to foaming; these reagents should be pipetted slowly. Careful handling of reagents is recommended to ensure the appropriate reagent is added at each step, and to prevent tube swapping. The correct magnet must always be used. For the standard workflow, a BLT-PF and TB1 master mix must not be made for tagmentation. If following the Hybex protocol with MIDI plates, changes to the workflow include use of a different magnet, shaking instead of pipetting, and difference in incubation step durations and temperatures due to plastic heat distribution.

Both workflows provide equivalent data.  The selection of the appropriate workflow is dependent upon the equipment available in the laboratory, and the training/familiarity associated with either approach by the end user.

Both single-stranded (ss) DNA Qubit and qPCR are suitable methods for library quantification, with further specific guidance found in the Reference Guide. ssQubit is recommended for standard DNA inputs (>300ng), whereas qPCR is recommended for low DNA input (< 300 ng). The conversion calculations for both methods will provide the double-stranded (ds) DNA molarity equivalent. Please note that quantification with dsDNA Qubit reagents will result in quantification failure.

RNA BioAnalyzer, Fragment Analyzer, and TapeStation cannot be used. The trace will not be visible.

Yield should be at least 2.8 nM by ssDNA Qubit for libraries prepared with >300ng input and at least 0.75nM by KAPA qPCR for libraries prepared with 100-299 ng input or when using the low input protocol.

Yes, the kit is designed to be automation compatible. Please see Illumina's Introduction to Sequencing Library Prep Automation for more details

 Illumina DNA PCR-Free, Tagmentation Library Prep is currently compatible with NovaSeq 6000.

Both strands of gDNA will be sequenced.

Libraries created with the standard input workflow can utilize the NovaSeq XP or Standard workflow. Libraries created with the low input workflow should utilize the NovaSeq XP workflow only. Loading concentrations for NovaSeq can be found in the Illumina DNA PCR-Free Reference Guide.

The kit components (with required storage temperatures -15C to -25C) have been validated for freeze-thaw up to 6 times.

Custom sequencing primers are required for Illumina DNA PCR-Free Library Prep. Custom sequencing primer boxes are sold on the library prep kit product page.

For those sequencing on NovaSeq (SBS v1), MiSeq and HiSeq 1000/1500/2000/2500, purchase Illumina® DNA PCR-Free Prep Sequencing Primers Read 1 (cat# 20041796). This kit contains 4 tubes of VP10 Custom Read 1 Primer (7.5 mL each).

For those sequencing on MiniSeq, NextSeq 500/550, and HiSeq 3000/4000, purchase Illumina® DNA PCR-Free Prep Sequencing Primers Read 1 + Index 2 (cat# 20041797). This kit contains 2 tubes of VP10 Custom Read 1 Primer (7.5 mL each) and 2 tubes of VP14 Custom Index 2 primer (10.5 mL each).

For NovaSeq reagents v1.0, follow the NovaSeq Series Custom Primers Guide.

For NovaSeq reagents v1.5, no custom primer kit is required. Followsd the guidance in the NovaSeq 6000 System Denature and Dilute Libraries Guide.

For all other platforms, refer to the Custom Primer Requirements for the Illumina DNA PCR-Free Prep, Tagmentation Kit bulletin, the instrument’s System Guide, and the instrument’s Denature & Dilute Guide.

 Library prep reagents, indexes, and SBS reagents are required for all preps. Most sequencing runs will also require custom sequencing primers. 

Additional equipment and consumables are also required depending on input amount, input type (gDNA, whole blood, saliva, dried blood spots), workflow type (thermal cycler or Hybex), and quantification method. Illumina DNA PCR-Free, Tagmentation libraries require quantification either by Kapa qPCR (any input amount) or with single-stranded Qubit reagents (input must be > 300 ng). Always consult the Reference Guide before beginning.

The IPB and AMPure XP beads differ slightly in properties, including sedimentation time. It may be possible to use AMPure beads, but the optimization of magnetic steps may be needed.