Illumina DNA PCR-Free FAQs

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  • Protocol

  • 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.

    For optimized index representation, Illumina recommends the use of IDT for Illumina DNA/RNA UD Indexes Set A, Tagmentation (20027213) or Illumina DNA/RNA UD Indexes Set B (20027214).

    To correct index pairs that underperform or overperform during library pooling, see the Balancing sample coverage for whole-genome sequencing  technical note for approximate expected index representations. Index correction is not supported by Illumina.

    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.