Impact of Pre-Analytical Variables on Cancer Targeted Gene Sequencing Efficiency.
Author(s): Araujo LH, Timmers C, Shilo K, Zhao W, Zhang J, Yu L, Natarajan TG, Miller CJ, Yilmaz AS, Liu T, Amann J, Lapa E Silva JR, Ferreira CG, Carbone DP
Publication: PLoS One, 2015, Vol. 10, Page e0143092
PubMed ID: 26605948 PubMed Review Paper? No
Purpose of Paper
This paper examined the effect that formalin-fixed paraffin-embedded (FFPE) block storage, DNA input amount and quality control (QC) parameters can have on the depth and efficiency of next generation sequencing results.
Conclusion of Paper
While the strength of correlations ranged from weak to moderate, longer FFPE storage durations, lower amounts of input DNA, and lower QC ratios were all significantly correlated to poor sequencing efficiency. The authors estimate that a sequencing depth of 50x coverage can be achieved for 90% of specimens if FFPE block storage is limited to 8.6 y or less, and thresholds of 0.22 for the PCR/QC ratio and 960 ng for DNA input amount are applied. Poor sequencing coverage was also correlated to low GC content (<0.4; p<0.01), a behavior that was mirrored in EGFR and KRAS hotspot codons.
Studies
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Study Purpose
This study examined the extent that FFPE block storage, DNA input amount and QC parameters can alter the depth and accuracy of next generation sequencing results. Tumors from 113 surgically resected lung specimens were analyzed, which included 110 primary non-small cell lung cancer (NSCLC) tumors and 3 head/neck tumors that had metastasized to the lung. Two to ten 10 µm-thick paraffin sections were macrodissected to maximize tumor content prior to DNA extraction with the Maxwell 16 FFPE Plus LEV DNA Purification kit. DNA quality was assessed with a PCR-based QC assay based on amplification of 105 bp and 236 bp amplicons, and a ratio of 0.2 was set as the quality threshold (favorable quality >0.2; moderate to poor quality ≤0.20) .
Summary of Findings:
Based on the PCR-based QC assay, approximately 50% of the 113 FFPE specimens yielded genomic (g)DNA of favorable quality (QC ratio > 0.02). Parameters of sequencing efficiency were correlated to varying degrees to FFPE block storage, PCR QC ratio and DNA input amount. Significant weak to modest negative correlations were observed for the duration for FFPE block storage and total number of reads (r= -0.356, p<0.01), mean target coverage (r= -0.405, p<0.01), alignment rate (r= -0.354, p<0.01), and mean base quality (r= -0.188, p=0.046), while a strong negative correlation was reported for insert size (r= - 0.764, p<0.01); conversely, a weak positive correlation was observed between FFPE block storage duration and the percentage of off-target reads(r=0.285, p<0.01). As expected, QC ratio was significantly, albeit modestly, correlated to insert size (r=0.601, p<0.01), and DNA input amount was modestly correlated to total number of reads (r=0.548, p<0.01), mean target coverage (r=0.549, p<0.01), alignment rate (r=0.449, p<0.01), mean base quality (r=0.477, p<0.01), but was weakly and negatively correlated to the percentage of off-target reads(r=-0.336, p<0.01). Based on multivariate analysis, the authors estimate that a sequencing depth of 50x coverage can be achieved for 90% of specimens if FFPE block storage is limited to 8.6 y or less, and thresholds of 0.22 for the PCR/QC ratio and 960 ng for DNA input amount are applied. The authors also observed poor coverage in regions with a low GC content (<0.4; p<0.01), a behavior was also observed in EGFR and KRAS hotspot codons. EGFR regions had optimal coverage and had a GC content of 0.51-0.55, while coverage of KRAS regions had much poorer coverage and GC content of 0.33-0.36.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA DNA sequencing DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0.32 - 24.22 y
DNA sequencing Specific Template/input amount 77-2337 ng
Real-time qPCR Specific Quality metrics 0.03-0.58 (PCR/QC ratio)