Top 20 FAQs on targeted NGS using QIAseq Targeted DNA Pro Panels
|Variant frequency (%)||Input (ng)||Read pairs/UMI||Mean read|
8. Can libraries from different panels be mixed and run together in the same run?
Yes. We removed the custom read 1 primer required in the Illumina sequencing process, which means users are now free to mix and match QIAseq Targeted DNA Pro libraries with other chemistries in the same run, even non-QIAseq ones.
9. Can you explain how the enzymatic clean up step works?
After ligation and target enrichment PCR, cleanup is carried out with an enzymatic reaction instead of beads. This dramatically reduces both hands-on and total time involved in the reaction cleanups. In addition, more consistent library construction can be achieved with the enzymatic cleanups as there’s no need for highly variable bead cleanups after the ligation and target enrichment PCR steps. This also makes the workflow more amenable to automation.
10. Does this completely eliminate bead cleanup steps?
No, this does not entirely eliminate bead cleanup steps. QIAseq Targeted DNA Pro Panels include just a single bead cleanup step after the Universal PCR step to purify the final libraries.
11. Is this workflow compatible with cfDNA samples?
Yes, the workflow is compatible with cfDNA samples.
12. For samples that are moderately compromised, do I need to use more DNA with this workflow than with high-quality samples?
The required amount of template for a single QIAseq Targeted DNA Pro sequencing reaction ranges from 10 to 80 ng for fresh DNA or cfDNA or 100 to 250 ng for formalin-fixed paraffin-embedded (FFPE) DNA.
13. What is the best sample QC method to use for the QIAseq Targeted DNA Pro workflow?
DNA integrity can be checked using the QIAxcel, Agilent Bioanalyzer or Agilent TapeStation. Although DNA is enzymatically fragmented before target enrichment PCR, intact DNA generally yields better results than fragmented DNA due to the tiling space between primers. Intact DNA usually has better coverage uniformity, more UMIs are captured and you achieve more sensitive variant detection. If FFPE DNA is used with QIAseq Targeted DNA Pro Panels, the QIAseq DNA QuantiMIZE Array or Assay Kit is strongly recommended for determining the optimal DNA amount for each FFPE DNA sample. Quantification based on mass calculations (OD, NanoDrop) cannot reliably measure the amplifiable amounts of DNA that are important for a multiplex PCR-based targeted enrichment NGS workflow, such as with the QIAseq Targeted DNA Pro Panels.
14. What software do you use to analyze your data?
We used QIAGEN CLC Genomics Workbench – QIAseq Targeted DNA Pro Plugin to analyze the data.
15. What is the upper limit of primers for custom panels?
The upper limit or a maximum number of primers for QIAseq Targeted DNA Pro Custom Panels is 20,000 primers.
16. Does QIAGEN provide a validated method script for Hamilton's line of STAR automated liquid handlers?
QIAseq Targeted DNA Pro Panels will have a validated automation script for Hamilton NGS STAR. Please work with your local Hamilton rep to install the script on your automation. QIAGEN will provide any additional technical support needed with the protocol.
17. Have you tested the panels at below 10 ng input? What is the expected performance at lower input? Would any analysis features likely be diminished (i.e., how low you can go with VAF)?
Yes, we observed consistent performance for specificity and uniformity across 1–80 ng of DNA input. However, we recommend 10–80 ng of DNA input (as per the kit handbook) for meaningful UMI capturing efficiency for calling low VAFs.
18. Do you expect fusion panels with the QIAseq Pro technology to be available in the coming future? If so, when?
Currently, QIAseq Pro technology, particularly the incorporation of enzymatic cleanups, is only applicable to targeted DNA-seq applications. You can analyze RNA fusions plus gene expression and SNVs in a single assay using our QIAseq Fusion XP Targeted Panels.
19. Do we need to re-design the primers when switching from QIAseq Targeted DNA Panels to QIAseq Targeted DNA Pro Panels?
To take full advantage of the QIAseq Targeted DNA Pro workflow, we need to ensure the primer pools are optimized for the QIAseq Targeted DNA Pro workflow to save you time and costs in the long run. Additionally, QIAseq Targeted DNA Pro technology uses GRCh38 for primer design to ensure the primer binding sites are consistent with the analysis in GRCh38. Some primers may need to be re-designed with appropriate primer binding sites in GRCh38 to make sure the reads are mapped accurately.
20. Is it possible to mix FFPE and cfDNA in the same run?
Yes, as long as they both have the same sequencing coverage requirement and you are following the recommended DNA input amount and DNA QC method for each sample type, you should be able to mix FFPE and cfDNA in the same run.