Frequently Asked Questions for Illumina FFPE DNA Prep with Exome 2.5 Enrichment

When to select this library preparation workflow? This kit is primarily designed to offer a tumor/normal workflow for Formalin-Fixed Paraffin-Embedded (FFPE) DNA samples for exome sequencing. Compared to Illumina DNA Prep with Exome 2.5 Enrichment, only Illumina FFPE DNA Prep with Exome 2.5 Enrichment is fully validated for exome sequencing using tumor/normal FFPE DNA input. It also offers additional benefits, including support for lower DNA input (40 ng), optional spike-in with the Twist Bioscience for Illumina Mitochondrial Panel or Illumina Custom Enrichment Panel v2, and the inclusion of unique molecular identifiers (UMIs), enabling sensitive variant detection down to 5% Variant Allele Frequency (VAF).

Input and Workflow

Should this kit be used for FFPE samples that are not tumor/normal matched?

If using non-tumor FFPE or high quality gDNA, the Illumina DNA Prep with Enrichment kit should be used instead.

Can this kit be used with other enrichment panels?

This workflow is only validated with the Twist Bioscience for Illumina Exome 2.5 Panel, with optional spike-in of the Twist Bioscience for Illumina Mitochondrial Panel or Illumina Custom Enrichment Panel v2.

How many freeze-thaw cycles are supported for reagents and index sets?

Reagents labeled for storage at −15°C to −25°C remain are supported for 6 freeze-thaw cycles per kit.

How should input be quantified and qualified?

Quantification should be performed with a fluorometric quantification method such as Qubit, while qualification should be performed with the TruSight FFPE QC kit. Use DNA samples that result in delta Cq (dCq) of ≤ 4.

Is there any guidance for using DIN or DV200 values instead of using the TruSight FFPE QC kit?

No, samples must be qualified by the TruSight FFPE QC kit.

Why is shearing needed when the input is already degraded?

It is for consistency. It is important to make sure all DNA is fragmented into sizes that can be incorporated into libraries.

Does this kit use tagmentation?

No, input DNA is sheared by Covaris. DNA is then end-repaired, A-tailed, and adapters are ligated, then the libraries are amplified prior to enrichment with the Twist Bioscience for Illumina Exome 2.5 panel. After enrichment, libraries undergo a final amplification and clean up prior to sequencing.

Is automation supported? If yes, how many samples can be processed per kit? Are there any Illumina-ready or known vendor-developed methods?

Yes, automation is supported. The kit name contains the number of samples that can be processed by automation. For example, the Illumina Cell-Free and FFPE DNA Prep, Ligation (16 Samples) (catalog 20104105) kit can process 16 samples by automation, or 24 sample manually. The number of samples that can be processed manually is provided in the product description on the product order page (select "View more" to expand product descriptions). There are no supported automation methods for Illumina FFPE DNA Prep with Exome 2.5 Enrichment kit at this time; Illumina is only providing automation-compatible reagents.

What are the supported plexities for enrichment?

Illumina only supports 4-plex enrichment with Illumina FFPE DNA Prep with Exome 2.5 Enrichment workflow.

Does the FFPE workflow also support the Twist Bioscience for Illumina Mitochondrial panel or Illumina Custom Enrichment Panel v2 designed via DesignStudio?

Yes, both supplemental enrichments are supported. Follow the guidance in the Supplemental Enrichment (Optional) section of the protocol.

Sequencing and Analysis

Which instruments and flow cell types does Illumina support for sequencing?

NovaSeq X/X Plus: 1.5B NovaSeq 6000: SP, S1, and S2 NextSeq 2000: P3 and P4 (XLEAP)

What is the recommended number of samples per run?

Similar information can also be found in the Denature and Dilute Protocol Generator. What is the targeted reads per sample to achieve 150X normal/500X tumor coverage.

28 million reads (clusters) for normal samples, and ~100 million reads (clusters) for tumor samples.

Is PhiX spike-in recommended?

Spike-in of PhiX is not required. However, PhiX spike-in can follow the general guidelines of spiking in 1-2% PhiX for additional sequencing metrics, if desired.

What is the recommended read length?

2x151 with 10 bp index reads.

What is the recommended loading concentration for all supported Illumina Platforms?

NovaSeq X/X Plus (1.5B only): 40 pM final loading concentration

• Note: One pool is loaded into both lanes to achieve sequencing depth requirements

NovaSeq 6000 (SP, S1, S2): 500 pM (0.5 nM) pooled library concentration; 100 pM final library concentration

• Note: Must use Standard loading so one pool goes into both lanes to achieve sequencing depth requirements

NextSeq 2000: 750 pM

Note: the above concentrations are starting guidelines; labs may need to optimize loading concentrations to maximize data yield and run performance.

Which analysis pipeline should be used for analysis and TMB (tumor mutational burden) calling?

DRAGEN Enrichment plus DRAGEN Somatic pipelines.

For any feedback or questions regarding this article (Illumina Knowledge Article #9485), contact Illumina Technical Support [e-mail].