Recommended quality control of FFPE samples for Illumina FFPE-supported library preparation kits
Formalin-fixed paraffin-embedded (FFPE) extraction methods generally yield highly degraded DNA and RNA, which presents a challenge for library preparation protocols. To determine whether FFPE samples are viable input material for downstream library preparation kit types, Illumina recommends performing FFPE quality control steps.
The following are FFPE-supported library preparation kits and their respective quality control steps.
DNA Sequencing - Whole Exome
Illumina DNA Prep with Enrichment (formerly Nextera Flex for Enrichment)
The ∆Cq value of the DNA sample can be assessed using the Infinium FFPE QC Kit (Illumina, WG-321-1001).
For FFPE samples with ∆Cq value of ≤ 5, the recommended DNA input is 50-1000 ng.
The Illumina DNA Prep with Enrichment kit is not recommended for use with samples with ∆Cq > 5.
Using samples with ∆Cq > 5 is possible but might increase chances of library preparation failure or decrease assay performance.
For extracted FFPE sample input, in the “Amplify Tagmented DNA” step of the library preparation protocol, increase PCR cycles to 12.
Targeted DNA Sequencing
AmpliSeq for Illumina Panels (Ready-To-Use)****: BRCA Panel, Cancer Hotspot Panel v2, Childhood Cancer Panel, Comprehensive Panel v3, Focus Panel, TCR beta-SR Panel, and Custom DNA Panels.
No FFPE Quality Control (QC) is required.
Do not exceed the maximum supported amount of input DNA.
Use 1 ng DNA only with high-quality, well-quantified samples.
No FFPE Quality Control (QC) is required.
Use at least 140 mm2 of nonmelanoma tissues with at least 30% tumor content.
Use a validated FFPE extraction kit:
AllPrep DNA/RNA FFPE Kit (QIAGEN)
QIAamp DSP DNA FFPE tissue Kit (QIAGEN)
ReliaPrep FFPE gDNA MiniPrep System (Promega)
The ∆Cq value of the DNA sample can be assessed using the Infinium FFPE QC Kit (Illumina, WG-321-1001).
For more information, refer to the FFPE DNA Extraction section of the TruSight Tumor 15 Reference Guide.
RNA Sequencing - Whole Transcriptome / Exome
Illumina Stranded Total RNA Prep with Ribo-Zero Plus
Use 10-100 ng FFPE RNA input with DV200 > 55%.
For more information, see the technical note Evaluating RNA Quality from FFPE Samples.
In the Amplify Library step of the library preparation protocol, increase PCR cycles by 2 for extracted FFPE sample input.
Assess FFPE RNA quality with the Bioanalyzer, Fragment Analyzer, or equivalent.
Adjust fragmentation time according to the Modify RNA Fragmentation Time for Degraded RNA section of the TruSeq Stranded Total RNA Sample Preparation Guide.
Adjust FFPE RNA input amount based on DV200 values. By adjusting RNA input amounts, high-quality libraries can be prepared from poor-quality FFPE samples.
For more information, see the technical note Evaluating RNA Quality from FFPE Samples.
Targeted RNA Sequencing - Fixed and Custom Panels
Illumina RNA Prep with Enrichment
Use 20-100 ng FFPE RNA input with DV200 ≥ 36.5%.
For more information, see the technical note Evaluating RNA Quality from FFPE Samples.
For extracted FFPE sample input, in the Tagment cDNA step of the library preparation protocol, increase PCR cycles to 17.
As for TruSeq RNA exome, adjust FFPE RNA input amount based on DV200 values.
For more information, see the technical note Evaluating RNA Quality from FFPE Samples.
Adjust FFPE RNA input amount based on DV200 values.
For more information, see the technical note Evaluating RNA Quality from FFPE Samples.
AmpliSeq for Illumina Panels: Transcriptome Human Gene Expression Panel, Immune Response Panel, Immune Repertoire Plus TCR beta Panel, and Custom RNA Panels.
Each reverse transcription reaction requires 1-100 ng of DNase-treated total RNA. The recommended input is 10 ng RNA.
Use 1 ng total RNA only with high-quality, well-quantified samples. Illumina recommends the use of Qubit to quantify input RNA. Do not use a UV-spectrometer-based method.
Library yield can be lower for degraded library samples such as FFPE RNA.
Targeted DNA/ RNA Sequencing - Fixed Panels
To obtain sufficient nucleic acid material, we recommend isolating nucleic acids from a minimum of 2 mm3 of FFPE tissue.
Input may need to be adjusted based on FFPE quality. For more information, please see the white paper Assessing DNA and RNA Quality from FFPE Samples for TruSight Tumor 170.
QIAGEN AllPrep DNA/RNA FFPE Kit is suggested for nucleic extraction.
DNA
Infinium FFPE QC Kit (Illumina, WG-321-1001) is recommended to check FFPE sample quality.
Recommend the use of DNA samples with ∆Cq value ≤ 5. Samples with a ∆Cq > 5 can decrease assay performance.
RNA
RNA sample quality can be assessed using Bioanalyzer, Fragment Analyzer, or equivalent.
Recommend the use of RNA samples that have a DV200 value of ≥ 20%. Using samples with a DV200 value < 20% may decrease assay performance.
TruSight Oncology 500 and TruSight Oncology 500 High Throughput (HT)
To obtain sufficient nucleic acid material, we recommend isolating nucleic acid from a minimum of 2 mm3 of FFPE tissue.
QIAGEN AllPrep DNA/RNA FFPE Kit is recommended for DNA extraction.
DNA samples can be assessed using the TruSight FFPE QC Kit (Illumina, 20139070).
Recommend use of DNA samples with ∆Cq value ≤ 5. Samples with a ∆Cq > 5 can decrease assay performance.
RNA samples can be assessed using Bioanalyzer, Fragment Analyzer, or equivalent.
Recommend use of RNA samples that result in a DV200 value of ≥ 20%. Using samples with a DV200 value < 20% may result in decreased assay performance.
The preceding sections summarize the quality control requirements and recommendations for use of FFPE samples with FFPE-supported library preparation kits. For more information, see the support page for each library preparation kit.
Quality control (QC) techniques mentioned in this article:
DV200 value is the percentage of RNA fragments > 200 nucleotides, calculated with the Bioanalyzer, Fragment Analyzer, or equivalent.
The Infinium FFPE QC kit (Illumina, WG-321-1001) is a qPCR-based measure that evaluates the quality of prospective DNA samples. 2x qPCR Master Mix containing green fluorescent dye must to be purchased separately from a general supplier.
The Infinium FFPE Restore kit is not used with the library prep kits listed above. This kit is only used for array assays to restore degraded FFPE DNA to an amplifiable state. This restore process is not compatible with sequencing applications.
For more information on FFPE DNA qualification, see the Infinium HD FFPE QC Assay Protocol.
TruSight FFPE QC Kit (Catalog# 20139070: uses a real-time PCR assay, targeting a 204 bp amplicon, to evaluate the quality of prospective DNA samples.
DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is degraded by the fixation process and can vary greatly in quality and usability for sequencing applications. The Illumina TruSight FFPE QC kit uses a real-time PCR assay targeting a 204 bp amplicon to evaluate the quality and suitability of prospective FFPE DNA samples for sequencing.
For any feedback or questions regarding this article (Illumina Knowledge Article #1941), contact Illumina Technical Support techsupport@illumina.com.
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