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)

• All the following are needed to assess sample quality:
• Infinium FFPE QC Kit (Illumina catalog number WG-321-1001) (user supplied)
• KAPA qPCR master mix (Universal) and Primer Premix (user supplied)
• Bio-Rad CFX96 Touch Real-Time PCR Detection System or equivalent instrument (user supplied)
• 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.

TruSeq DNA Exome

• No FFPE QC is required.
• Adjust FFPE DNA input based on Genomic Quality Number (GQN).
• To increase library yield for samples with GQN values ≥ 0.3, increase PCR cycles in the first amplification of the protocol to 12 cycles.
• Increase the amount of each library used for enrichment to 500 ng regardless of initial input amount.
• For more information regarding the previous recommendations, refer to the technical note Evaluating DNA Quality from FFPE Samples.

Targeted DNA Sequencing

AmpliSeq for Illumina Panels (Ready-To-Use): BRCA Panel, Cancer Hotspot Panel v2, Childhood Cancer Panel, Comprehensive Panel v3, Comprehensive Cancer Panel, Focus Panel, and TCR beta-SR Panel; AmpliSeq for Illumina Custom DNA Panel (custom panel)

• No FFPE QC is required for the AmpliSeq kits.
• Do not exceed the maximum supported amount of input DNA.
• Use 1 ng DNA only with high-quality, well-quantified samples.

TruSight Tumor 15

• 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)
• 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 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.

TruSeq Stranded Total RNA

• Assess FFPE RNA quality by 2100 Bioanalyzer Desktop System (Agilent Technologies, part # G2940CA) with Agilent RNA 6000 Nano Kit (part # 5067-1511).
• Or assess FFPE RNA quality by Fragment Analyzer Automated CE System (Advanced Analytical Technologies, part # FSv2-CE2 or FSv2-CE10) with Standard Sensitivity RNA Analysis Kit (20 nt Lower Marker) (Advanced Analytical Technologies, part # DNF-489).
• Adjust fragmentation time according to the Modify RNA Fragmentation Time for Degraded RNA section of the TruSeq Stranded Total RNA Sample Preparation Guide.

TruSeq RNA Exome

• 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.

TruSight Pan Cancer

• 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.

TruSight RNA Fusion

• 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, and AmpliSeq for Illumina Custom RNA Panel

• 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

TruSight Tumor 170

• 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
• Illumina FFPE QC kit (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 Advanced Analytical Technologies Fragment Analyzer (Standard Sensitivity RNA Analysis Kit) or Agilent Technologies 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit).
• 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 Illumina FFPE QC kit (WG-321-1001).
• Recommend use of DNA samples with ∆Cq value ≤ 5. Samples with a ∆Cq > 5 can decrease assay performance.

• RNA samples can be assessed using Advanced Analytical Technologies Fragment Analyzer™ (Standard Sensitivity RNA Analysis Kit) or Agilent Technologies 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit).
  • Recommend use of RNA samples that result in a DV200 value of ≥ 20%. Using samples with a DV200 value < 20% might 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 Bulletin:

• DV200 value is the percentage of RNA fragments > 200 nucleotides, calculated with the Bioanalyzer or the Fragment Analyzer.
• The Infinium FFPE QC kit is a qPCR-based measure that evaluates the quality of prospective DNA samples.
  • 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.
  • For more information on FFPE DNA qualification, see the Infinium HD FFPE QC Assay Protocol.