Formalin-fixed, paraffin-embedded (FFPE) samples are preserved tissue samples commonly used to investigate disease pathology. FFPE extraction methods generally yield highly degraded DNA and RNA, which presents a challenge for library preparation protocols. To determine if FFPE samples are viable input material for downstream library preparation kit types, we recommend certain FFPE quality control steps.

FFPE-supported library preparation kits and their respective quality control steps:

 

DNA Sequencing – Whole Exome

TruSeq Exome

• No FFPE QC kit required
• FFPE optimization recommendations for the TruSeq Exome kit:
  • Vary FFPE DNA input based on Genomic Quality Number (GQN)
  • Increase PCR cycles in the first amplification to 12 for samples with GQN values > 0.3 to increase library yield
• Increase the amount for enrichment to 500 ng regardless of initial input amount
• For more information, see the technical note Evaluating DNA Quality from FFPE Samples

 

Targeted DNA Sequencing – Fixed Panel

TruSeq Amplicon Cancer Panel

• Illumina FFPE QC kit (WG-321-1001) is required to check FFPE sample quality
• For more information, see the guide for Illumina FFPE QC Kit for TruSeq Amplicon - Cancer Panel and TruSeq Custom Amplicon v1.5

TruSight Tumor 15

• No FFPE QC kit required
• For FFPE samples, adhere to the following extraction recommendations:
  • Use at least 140 mm² of nonmelanoma tissues with at least 30% tumor content
  • Use validated extraction kits:
    • AllPrep DNA/RNA FFPE kit (QIAGEN)
    • QIAamp DSP DNA FFPE tissue kit (QIAGEN)
    • ReliaPrep FFPE gDNA MiniPrep System (Promega)
• For more information, see the FFPE DNA Extraction section of the TruSight Tumor 15 Reference Guide

TruSight Tumor 26

• No FFPE QC kit required
• Extract DNA using the QIAGEN Supplementary Protocol: Purification of Genomic DNA from FFPE tissue using the QIAamp DNA FFPE tissue kit and Deparaffinization Solution [Refer to QIAGEN resource website for protocol]
• To determine the amplification potential and input amount, see the Qualify DNA Extracted from FFPE Samples section in the TruSight Tumor 26 Reference Guide
• For more information, see the white paper Generating Sequencing Libraries from FFPE Samples

 

Targeted DNA Sequencing – Custom Panel

TruSeq Custom Amplicon v1.5

• Illumina FFPE QC kit (WG-321-1001) is required to check FFPE sample quality
• For FFPE samples, supported amplicon size for design is only 150 bp and 175 bp. The FFPE input criteria is a minimum of 250 ng with A260/A280 = 1.8–2.0
• For more information, see Illumina FFPE QC Kit for TruSeq Amplicon - Cancer Panel and TruSeq Custom Amplicon v1.5

TruSeq Custom Amplicon Low Input

• TruSeq FFPE DNA QC kit (FC-121-9999) is required to check FFPE sample quality
• Supported amplicon size for FFPE designs is only 150 bp and 175 bp, dual pool design is recommended
• For more information, see the TruSeq FFPE DNA Library Prep QC Reference Guide

 

RNA Sequencing

TruSeq Stranded Total RNA

• Assess FFPE RNA quality by Bioanalyzer
• Vary fragmentation time according to the Modify RNA Fragmentation Time for Degraded RNA section of the TruSeq Stranded Total RNA Sample Preparation Guide

TruSeq RNA Access

• Vary FFPE RNA input based on DV200 values
• For more information, see the technical note Evaluating RNA Quality from FFPE Samples

 

Targeted RNA Sequencing – Fixed Panels

TruSight Pan Cancer

• Vary FFPE RNA input based on DV200 values
• For more information, see the technical note Evaluating RNA Quality from FFPE Samples

TruSight RNA Fusion

• Vary FFPE RNA input based on DV200 values
• For more information, see the technical note Evaluating RNA Quality from FFPE Samples

TruSeq Targeted RNA Expression p53, Wnt, NFkB, Stem cell, Hedgehog, Cell cycle, Apoptosis Panels

• Use 100-200ng degraded or FFPE samples with an average fragment size of >200 bp for optimal hybridization. In addition, check the RNA for total RNA integrity number (RIN) following isolation on an Agilent Technologies 2100 Bioanalyzer using an RNA 6000 Nano Chip.
• For more information, see the RNA Input Recommendations section of the TruSeq Targeted RNA Expression Reference Guide
• For more information, see the technical note Expression Analysis of FFPE Samples

 

The above summarizes the quality control requirements and recommendations for the use of FFPE samples for supported library preparation kits. For more information, see the support page for each library preparation kit.