Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples.
Author(s): Arreaza G, Qiu P, Pang L, Albright A, Hong LZ, Marton MJ, Levitan D
Publication: Int J Mol Sci, 2016, Vol. 17, Page
PubMed ID: 27657050 PubMed Review Paper? No
Purpose of Paper
This paper compared DNA yields by two different extraction methods and amplifiability by three methods of formalin-fixed paraffin-embedded (FFPE) tumor specimen sections by four different laboratories. The effect of adjusting DNA input based on amplifiability on the whole exome sequencing library complexity was also examined.
Conclusion of Paper
DNA yield and amplifiability varied between laboratories, even when the same extraction kit was used but DNA yield was higher when extracted using QIAamp rather than RecoverAll. DNA yields were generally 2-10 fold higher by Qubit than by spectrophotometry and there was more consistency between labs when DNA was quantified by Qubit. Relative amplifiability differed between the various assays. Adjusting the DNA input based on the Assuragen Quantitative Functional Index (QFI) increased the pre-capture PCR yield and post-capture PCR library concentrations for specimens with pre-capture PCR yield and post capture PCR library concentrations below in the cut-off values so they passed quality control.
Studies
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Study Purpose
This study compared DNA yields by two different extraction methods and amplifiability by three methods of formalin-fixed paraffin-embedded (FFPE) tumor specimen sections by four different laboratories. The effect of adjusting DNA input based on amplifiability on the whole exome sequencing library complexity was also examined. Twenty archival FFPE blocks containing colorectal cancer, renal cell carcinoma, breast cancer, or lung cancer that had been stored for 1-5 years were freshly sectioned (5µm) and placed on slides. Five slides from each block were sent to each of four commercial labs for DNA extraction. DNA was quantified in the laboratory that performed extraction by their method of choice and again by Qubit and Nanodrop after shipment back to the central laboratory.
Summary of Findings:
The yield of DNA as determined by Qubit varied significantly among the laboratories with differences of up to 5-10-fold occurring between the three labs that used the QIAamp FFPE kit and the lab that used the RecoverAll kit and up to a 2-3-fold difference in yield between labs using the same kit. Amplifiability of DNA extracted from the lab using RecoverAll kit was highly variable between specimens, but there was much less variability between labs and between specimens when DNA was extracted with QIAamp. Similarly, there was a lot of inter-lab and inter-kit differences in the amount of amplifiable DNA when quantified in the central laboratory using the QuantideX qPCR DNA QC Assay and the RNase P assay. Interestingly, relative amplifiability was not similar between assays. Adjusting the DNA input based on QFI increased the pre-capture PCR yield and post-capture PCR library concentrations so they passed quality control in specimens with pre-capture PCR yield and post capture PCR library concentrations below the cut-off values. DNA yields were generally 2 to 10-fold higher and much more variable when quantified by spectrophotometry than Qubit. The authors report that Qubit quantification shows good correlation with droplet digital PCR.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Real-time qPCR DNA Next generation sequencing DNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp FFPE kit (at 3 laboratories)
RecoverAll
Fluorometry Specific Technology platform Nanodrop spectrophotometry
Next generation sequencing Specific Template/input amount 50 ng
Adjusted based on QFI
Real-time qPCR Specific Targeted nucleic acid Unspecified
TBP
RPPH1
Real-time qPCR Specific Technology platform Varied in-house protocols
QuantideX qPCR DNA QC Assay
RNase P assay