Table 1.
Pre-analytical parameters and sequencing metrics.
Fig 1.
Three pre-analytical variables (FFPE storage time, PCR/QC ratio, and DNA input in the library preparation) were significantly correlated to most post-sequencing parameters (A). The pre-analytical variables were classified as below or above median values to illustrate the impact on insert size (B) and on read quality/Phred score (C). Abbreviations: FFPE, formalin-fixed paraffin-embedded tissue blocks; PCR/QC, PCR-based quality control.
Fig 2.
FFPE storage time (A), PCR/QC ratio (B), and DNA input (C) were correlated to sequencing depth of coverage. A combined score (D) was constructed based on these three parameters, and was highly correlated to sequencing depth. Abbreviations: FFPE, formalin-fixed paraffin-embedded tissue blocks; PCR/QC, PCR-based quality control.
Fig 3.
The combined score was strongly correlated to post-sequencing parameters (A). Correlation to the 50x coverage was used to define pre-analytical thresholds that could predict sequencing efficiency, and are illustrated by smoothing curves (B).
Table 2.
Summary of correlation analyses between the pre-sequencing combined score and final sequencing parameters.
Fig 4.
Normalized depth of coverage presents wide variability within the genome, with worse coverage observed in regions with lower GC content (A). The GC content effect was additive to sample quality to predict depth of coverage, as observed after stratifying sample quality using the combined score (B). Abbreviations: St. Dev., standard deviation. Obs: ** indicates significance at p<0.01.
Table 3.
GC content and depth of coverage at hotspot positions for recurrent somatic mutations in non-small cell lung cancer.