FFPE DNA Quality: Impact on Downstream Performance in Molecular Diagnostic AssaysFFPE DNA Quality: Impact on Downstream Performance in Molecular Diagnostic AssaysJonathan M. Eibl, Julie A. Toplin, Chad D. Galderisi, Cindy S. Spittle, MolecularMD Corp., Portland, OR
IntroductionPersonalized medicine for oncology patients is driven by mutation profiling of tumors.Improved robust DNA extraction methods that produce high quality FFPE DNA template
Poster #: ID102
Figure 3a. Maxwell vs. MO BIO DNA extraction from different FFPE sample types (total DNA Yield). Overall, DNA yields were greater for
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Figure 3b. Percent increase in DNA yield by Maxwell vs. MO BIO in different FFPE sample types (1.9‐fold or 190% increase overall).
Figure 6a. Box plot graphs of Phred scores for Sanger sequences (see Figure 5; forward traces; 51‐bp window; melanoma samples).
Figure 6b. Box plot graph of Phred scores for Sanger sequences (see Figure 5; reverse traces; 51‐bp window; melanoma samples). p p g q y p
suitable for complex molecular diagnostic testing are needed. In addition to DNAfragmentation and protein‐DNA cross linking, some FFPE tissues contain inhibitors and/orquenching substances (e.g. melanin) that adversely affect downstream performance in PCRand sequencing assays. In this study, we provide a side‐by‐side evaluation of two DNAisolation methods from FFPE tissue of various tumor types: a manual, column‐basedmethod (MO BIO) and the Maxwell CSC automated platform (Maxwell). 40
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Materials & MethodsFFPE tissue from melanoma, GIST, endometrial, and NSCLC tumor types was obtained fromequivalent numbers of slides for each extraction method. Manual extractions wereperformed using the MO BIO BiOstic FFPE Tissue DNA Isolation Kit followed in some
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performed using the MO BIO BiOstic FFPE Tissue DNA Isolation Kit followed in someinstances by an additional purification step using the Zymo Research OneStep PCR InhibitorRemoval Kit to rid DNA isolates of visible melanin. Automated extractions were carried outusing the Maxwell CSC DNA FFPE Custom Kit on the Maxwell CSC instrument with noadditional PCR inhibitor removal steps. DNA eluate yields and optical densities weredetermined via spectrophotometry. Equivalent input DNA amounts from melanomasamples were assayed using both NRAS allele‐specific qPCR (25 ng) and NRAS Sanger
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Melanoma GIST Endometrial NSCLC
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Melanoma GIST Endometrial NSCLC
samples were assayed using both NRAS allele specific qPCR (25 ng) and NRAS Sangersequencing (200 ng) on the BIORAD CFX platform and Applied Biosystems 3730 DNAAnalyzer, respectively. NRAS qPCR data were reported as delta‐Ct values to determine qPCRdifferences. Sanger sequencing Phred scores were analyzed for the 51‐base pair windowsurrounding the NRAS codon 61 hotspot to quantitatively determine sequence‐trace quality.Quality metrics were further evaluated using the CodonCode Aligner and Sequence Scannersoftware packages 3
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4OD 260/280 MaxwellOD 260/280 MO BIO 2.5
3OD 260/230 MaxwellOD 260/230 MO BIO
Figure 4a. Optical densities (260/280) Maxwell vs. MO BIO DNA extraction from different FFPE sample types.
Figure 4b. Optical densities (260/230) Maxwell vs. MO BIO DNA extraction from different FFPE sample types.
Figures 7a – 7e. Box plots representing sequence quality metrics using CodeCodon Aligner and Sequence Scanner software.
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ResultsBoth extraction methods require the same upfront steps of scraping FFPE tissue, reagentaddition, and lysis/incubation, however, the Maxwell method requires fewer hands‐on,liq id handling steps (Fig 1) Additional Z mo clean p steps ere req ired for 2 man all
software packages.
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liquid‐handling steps (Fig. 1). Additional Zymo cleanup steps were required for 2 manually‐extracted samples due to visible melanin in the FFPE tissue/eluted DNA (Fig. 2; Samples 02and 07). DNA yields were greater for samples processed on the Maxwell CSC (30 of 34 totalsamples, 88%; 1.9‐fold increase overall; Figs. 3a and 3b). Optical densities for both methodswere largely congruent for the 260/280 ratio (Fig. 4a) but more variable for the 260/230ratio (Fig. 4b). In qPCR, 11 of 12 (92%) of the Maxwell‐extracted melanoma samples crossedthe threshold 1 3 Ct sooner on a erage than the corresponding man all e tracted
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Melanoma GIST Endometrial NSCLC
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Melanoma GIST Endometrial NSCLC
Figure 5a NRAS allele‐specific PCR for DNA extracted by both methods Figure 5b Comparison of NRAS Sanger sequencing results for DNA extracted by
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the threshold 1.3 Ct sooner on average than the corresponding manually‐extractedtemplates (Fig. 5a), and one manually‐extracted, Zymo‐purified sample failed to amplify(Sample 07). Two Maxwell‐extracted samples (Samples 10 and 11) crossed the thresholdmarkedly sooner (~2.5 Ct). Similarly, all Maxwell‐extracted melanoma DNA isolatesproduced robust bidirectional sequence, whereas two manually‐extracted isolates failed toyield NRAS sequence despite the inhibitor‐removal step (Fig. 5b). Aside from the sequencef il f th t l t d dditi l t l t hi h hibit d l t d b li
Sample Ct Value ∆ Ct Value (MO BIO – Maxwell)
MO BIO 01 33.041.11
Maxwell 01 31.93
MO BIO 02Maxwell 02
Figure 5a. NRAS allele‐specific PCR for DNA extracted by both methods from melanoma FFPE; traces shown at right for samples with ∆Ct Values ˃ 2.0 (Samples 02, 10, 11) and no amplification/amplification (Sample 07).
Figure 5b. Comparison of NRAS Sanger sequencing results for DNA extracted by both methods from two melanoma samples, both of which required the Zymocleanup step for the MO BIO extraction.
failures of these templates and one additional template which exhibited elevated baselinenoise, NRAS codon 61 mutation‐detection results for the remaining nine manually‐extractedeluates were identical to the Maxwell‐extracted samples (data not shown). Templates fromboth extraction methods exhibited higher median Phred scores and narrower interquartileranges (IQR) for reverse traces than for forward traces (Figs. 6a and 6b); this characteristic isattributed to the proximal location of the forward primer relative to the NRAS codon 61h t t S f M ll t t d FFPE DNA hibit hi h Ph d
MO BIO 02 35.422.17
Maxwell 02 33.25MO BIO 03 29.93
0.37Maxwell 03 29.56MO BIO 04 32.09
0.66Maxwell 04 31.43MO BIO 05 32.50
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hotspot. Sequences from Maxwell‐extracted FFPE DNA exhibit higher mean Phred scoresand less variability (insets; Figs. 6a and 6b). Overall, metrics generated using other softwarepackages (i.e., quality values, trace scores, quality value 20+, continuous‐read length, andsignal‐to‐noise ratio) illustrate equivalent or better results for the Maxwell‐extractedtemplates especially in regards to variability (see IQRs for boxplots; Figs. 7a –7e).
Conclusions
0.73Maxwell 05 31.77MO BIO 06 34.64
1.94Maxwell 06 32.70MO BIO 07 No amplification
N/AMaxwell 07 35.07MO BIO 08 30.10
‐0.02Maxwell 08 30.12
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This comparison highlights the potential advantages of using an automated nucleic acid extraction platform for onco‐diagnostictesting:
• Standardized workflow with fewer hands‐on steps minimizing the likelihood of cross‐contamination
• Higher throughput for faster turn‐around‐time of results
i h i ld i i i h i l d f li i d f i
Figure 1. Comparison of extraction method procedural steps and hands‐on time
Scrape FFPE Add reagents Lyse (incubate)
Steps common to both extraction methods
MO BIO 09 30.111.72
Maxwell 09 28.39MO BIO 10 30.93
2.60Maxwell 10 28.33MO BIO 11 30.80
2.55Maxwell 11 28.25MO BIO 12 28.44
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• Higher DNA yields, maximizing the potential data output from limited amounts of FFPE tumor tissue
• Higher quality DNA without extra purification steps, minimizing the potential need for repeat analysisMaxwell: post‐lysis steps 1 – 12 (Hands‐on time for 16 samples ‐ ~30 min.)
Cool to RT Add RNase A
Incubate (5 min.)
Place cartridges
Remove seals
Place plungers
Place elution tubes
Add elution water
Spin FFPE digest (5 min.)
Add sample to cartridge
Enter sample
data / run
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Figure 2. Representative specimen (Sample 02) requiring additional Zymo cleanup steps due to high melanin content (note red boxed area); H&E staining, 4X magnification.
0.05Maxwell 12 28.39
AcknowledgementsThe authors would like to thank Jeannette Nussbaum for her valuable contributions
For further information please contact [email protected] or visit www.molecularmd.com. MO BIO: post‐lysis steps 1 – 22 (Hands‐on time for 16 samples ‐ ~60 min.)
Heat (1 hr.)
Spin (1 min.) Transfer Add FP4 Vortex Add FP5 Vortex Spin Apply to
columnSpin
(1 min.)Transfer spin filter
Add FP6 (continue)
Transfer column
Spin (1 min.)
Add FP7 (1 of 2)
Spin (1 min.)
Add FP7 (2 of 2)
Spin (1 min.)
Spin (2 min.) Add FP8 Incubate
(5 min.)Spin
(1 min.)
ZYMO cleanup
(as needed)
*
The authors would like to thank Jeannette Nussbaum for her valuable contributions.
*(28 total steps if Zymo needed)
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