Effects of Improved DNA Integrity by Punch From Tissue Blocks as Compared to Pinpoint Extraction From Unstained Slides on Next-Generation Sequencing Quality Metrics.
Author(s): Morlote D, Janowski KM, Siniard RC, Guo RJ, Winokur T, DeFrank G, Harada S
Publication: Am J Clin Pathol, 2019, Vol. , Page
PubMed ID: 30892602 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of harvesting tissue from formalin-fixed paraffin embedded (FFPE) specimens using a punch rather than Pinpoint method on DNA yields and integrity and Next-Generation Sequencing (NGS) metrics. The effects of block age and specimen type (biopsy versus excision) on DNA integrity were also investigated.
Conclusion of Paper
The average concentration of DNA and DNA integrity numbers (DIN) were significantly higher when the specimen was harvested using the punch rather than the Pinpoint method. Although the average age of the FFPE blocks was non-significantly older for Pinpoint than punch specimens and there was a weakly negative correlation between block age and DIN, the DIN was still significantly lower in Pinpoint specimens than punch specimens after adjustment of the DIN for block storage. DINs were comparable between biopsy and excision specimens, regardless of harvesting method. Punch specimens had a lower percentage mapped on-target reads but higher percentage unique on-target reads, average coverage, and coverage at 100X, 400X, and 1000X than Pinpoint specimens, regardless of block age. DIN was found to correlate with percentage of unique reads on-target, average coverage, and positions with greater than 100X, 400X, and 1000X coverage. When specimens were stratified by DIN, the average percentage unique on-target was significantly higher in specimens with higher DIN.
Studies
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Study Purpose
The study investigated the effects of harvesting tissue from FFPE specimens using punch rather than Pinpoint on the DNA yields and integrity and NGS metrics. The effects of block age and specimen type (biopsy versus excision) on DNA integrity were also investigated. The study included 199 routine clinical biopsy and excision FFPE colorectal adenocarcinoma, lung adenocarcinoma, melanoma, and glioblastoma specimens. Details of specimen procurement and processing including fixation duration were not specified but, for each specimen, an area of interest (preferable >20% neoplastic, but at least 10%) was identified by a molecular pathologist based on an H&E stained slide. For the 76 specimens obtained between September 2016 and February 2017, DNA was extracted from ten 7-10 µm sections using the Pinpoint slide DNA isolation system. The sections were deparaffinized in dewaxing solution and Pinpoint solution was applied to a pathologist selected area. The solution was allowed to dry at room temperature for 30-45 min and sections were lifted from the slide and then placed in a tube with extraction buffer containing proteinase K. The tube was incubated with mixing at 55˚C for 4 h to overnight. RNAse A was added to the lysate and the genomic DNA was extracted using the QIAamp kit and a QIAcube. For the 123 specimens collected after February 2017, 1-3 tissue cores obtained with a 1 mm disposable Integra Miltex biopsy punch were deparaffinized with Qiagen Deparaffinization solution at 55˚C for 30 min. The deparaffinization solution was removed by centrifugation, Qiagen ATL lysis buffer containing proteinase K was added, and the specimen was incubated at 56˚C overnight followed by at 90˚C. The lysate was treated with RNAse A and DNA was extracted using the QIAamp kit and a QIAcube. DNA concentration was determined using Qubit fluorometry and the purity by spectrophotometry. DNA integrity was determined using DNA ScreenTape. DNA was hybridization-based target enriched for 90 genes using the Agilent HaloPlex HS sequencing method. DNA was sequenced using an Illumina MiSeq instrument.
Summary of Findings:
Although the elution volume was the same, the average and median concentration of DNA were significantly higher when the specimen was harvested using the punch (130.6 ng/µL and 88.10 ng/µL, respectively) rather than the Pinpoint method (36.83 ng/µL and 20 ng/µL, respectively, P<0.001). The average and median DNA integrity numbers (DIN) were also higher for specimens obtained by the punch method (6.18 and 6.30, respectively) than the Pinpoint method (5.09 and 5.40, respectively, P<0.001); however, the authors noted the average age of the FFPE blocks was non-significantly older for Pinpoint than punch specimens (average of 135.89 versus 86.05, P=0.2503) and there was a weakly negative correlation between block age and DIN (r=-0.35, P<0.001). After adjustment of the DIN for block storage, the DIN was still significantly lower in Pinpoint specimens than punch specimens. DINs were comparable between biopsy and excision specimens regardless of extraction method, but the average DIN for each was higher when extracted using the punch method (P<0.0001, both).
Punch specimens had a significantly lower percentage mapped on-target reads, but significantly higher percentage unique on-target reads than Pinpoint, regardless of block age (0-20 days or >20 days, P<0.0001, all). Further, the average coverage and percentage of positions with over 100X, 400X, and 1000X coverage were all higher for punch than Pinpoint specimens, regardless of block age (P<0.0001, all). DIN was found to correlate with percentage of unique reads on-target (r=0.60, P<0.0001), average coverage (r=0.57, P<0.0001), and positions with greater than 100X (r=0.62, P<0.0001), 400X (r=0.60, P<0.0001) and 1000X coverage (r=0.55, P<0.0001). When specimens were stratified by DIN, the average percentage unique on-target was higher in specimens with higher DIN (64.45% for DIN ≥ 7.0, 49.10% for DIN 4.0-6.9, and 32.60% for DIN 1.0-3.9; P<0.0001).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Mixed type
- Neoplastic - Melanoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Spectrophotometry DNA Next generation sequencing DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Method of tissue acquisition Biopsy
Surgical resection
Storage Storage duration 0-20 days
>20 days
Biospecimen Aliquots and Components Aliquot size/volume Section
Punch