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
TruSight
RNA Fusion
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.