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 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 use with FFPE samples for exome sequencing. Additional benefits include supporting a lower input amount (40 ng), optional spike-in with the Twist Bioscience for Illumina Mitochondrial Panel or Illumina Custom Enrichment Panel v2, and includes UMIs, which allow for sensitive variant detection down to 5% VAF.
While Illumina DNA Prep with Enrichment has been tested with FFPE samples, Illumina FFPE DNA Prep with Exome 2.5 Enrichment offers additional benefits of full validation with tumor/normal FFPE DNA input, supports a lower input amount (40 ng), optional spike-in with the Twist Bioscience for Illumina Mitochondrial Panel or Illumina Custom Enrichment Panel v2, and includes UMIs, which allow for sensitive variant detection down to 5% 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 new FFPE kit be used with other enrichment panels?**This workflow is only validated with the Twist Exome 2.5 enrichment panel, with optional spike-in of the Twist Bioscience for Illumina Mitochondrial Panel or Illumina Custom Enrichment Panel v2.
**How many freeze-thaws do reagents and index sets support?**Reagents with label storage temperatures from -15°C to -25°C will remain functional for at least 6 kit freeze-thaw cycles.
**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 pieces 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 and amplified prior to enrichment with the Twist Bioscience for Illumina Exome 2.5 panel. Libraries go through 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 Qualified 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 contained in the "i" information dropdown on the product page.
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.
**What are typically pre-enrichment library yields and final library yields?**Pre-enrichment library yield is expected to be > 150 ng/µl. Final library yield is expected to be > 3 ng/µl.
Sequencing and Analysis****What instruments does Illumina support for sequencing?
NovaSeq X: 1.5 B
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. Is it 28M reads to normal and ~72M reads to tumor? (M = million)**28M for Normal, and ~100M for Tumor single-end reads.
**Is PhiX spike in recommended?**Spike-in of PhiX is not required. However, customers 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?
NextSeq 2000: 750 pM
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
NovaSeq X (1.5B only): 40 pM final loading concentration
Note: One pool goes into both lanes to achieve sequencing depth requirements
**Which analysis pipeline should be used for analysis and TMB (tumor mutational burden) calling?**DRAGEN Enrichment plus DRAGEN Somatic pipelines.
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