A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single- Nucleotide Variant Detection Chung Lee, Joon S. Bae, Gyu H. Ryu, Nayoung.

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Presentation transcript:

A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single- Nucleotide Variant Detection Chung Lee, Joon S. Bae, Gyu H. Ryu, Nayoung K.D. Kim, Donghyun Park, Jongsuk Chung, Sungkyu Kyung, Je-Gun Joung, Hyun-Tae Shin, Seung-Ho Shin, Younglan Kim, Byung S. Kim, Hojun Lee, Kyoung-Mee Kim, Jung-Sun Kim, Woong-Yang Park, Dae-Soon Son The Journal of Molecular Diagnostics Volume 19, Issue 5, Pages 651-658 (September 2017) DOI: 10.1016/j.jmoldx.2017.06.001 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Theoretical detection probabilities according to variant allele frequency. Theoretical detection probabilities were calculated using a cumulative binomial distribution. When a 1% false positive (type I error) is allowed, variants of 9.2% (dashed line, left) at 200× and 3.8% (dashed line, right) at 500× can be detected. The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Comparison of the suggested pass rate (PR) score, uniformity, and mean depth. PR scores of the fresh-frozen (A) and formalin-fixed, paraffin-embedded (B) samples are dotted by graded color. The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Discordant example of the pass rate (PR) score with mean depth and uniformity. Visualization of the distribution of 167 hotspots used in the calculation of PR scores for the two samples analyzed in Supplemental Figure S1. Each gene was divided into different colors, and detected mutations were marked with a bold box with its amino acid change. A: Case 1: Although a relatively low mean depth (324×) is produced, there is high uniformity (0.938) and a perfect PR score (PR200 = 100%). B: Case 2: High mean depth (634×), low uniformity (0.371), and low PR score. Many hotspots are distributed under 200× (dashed lines). The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Classification of quality control (QC) success and failure based on experimental factors. Regression analysis results are shown for each of the experimental factors to identify the parameters affecting QC success or failure. Fresh-frozen (FF; A) and formalin-fixed, paraffin-embedded (FFPE; B) models used pass rate (PR200 and PR500) scores as response variables, respectively. A PR score cutoff of 80 was used as the binary variable to determine success/failure. The number in a box indicates the number of samples for success and failure, and the rate of sample success as following the box with an asterisk. Thirty-eight FF samples and three FFPE samples were lost because of missing records. N, no; Y, yes. The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Relationship between the pass rate (PR) score and the amount of data output with respect to the amount of DNA input. PR scores were calculated through in silico down-sampling from a total of 2.5 million reads up to 63.3 million in a pooled set of 10 HapMap cell lines (XX sample). DNA input amounts were determined from 250 down to 6.25 ng by serial dilution. Sequencing was repeated in triplicate. The last point (right side) of each input amount corresponds to the original sequencing data produced. The other points indicate the PR score that was calculated using the in silico down-sampled data. Data are expressed as means ± SEM. The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Discordant example of the pass rate (PR) score with mean depth and uniformity. A: Case 1: Although a relatively low mean depth (324×) is produced, there is high uniformity (0.938) and a perfect PR score (PR200 = 100%). B: Case 2: High mean depth (634×), low uniformity (0.371), and low PR score. Many hotspots are distributed under 200× (horizontal dashed lines). Vertical dotted lines differentiate each chromosome in ascending order (leftmost to rightmost). The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Comparison of the depth distribution between two genomic DNA extraction kits. Adjacent normal stomach and colon formalin-fixed, paraffin-embedded tissues were used to compare the two kits. Sequencing was performed after genomic DNA extraction using the two kits (conducted in duplicate), and the mean depth of each exon in the target region was plotted. Horizontal dashed lines indicate 200× coverage. Vertical dotted lines differentiate each chromosome in ascending order. The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Relationship between pass rate (PR) score and performance measure. A and B: We compared performance measures [detection sensitivity (A) and positive predictive value (B)] and PR score by in silico down-sampling using XX cell line mix and formalin-fixed, paraffin-embedded (FFPE) sample data, which are used in our previous study.15 Setting of down-sampling and definition of performance measures were used as the same. C: The proportion of chromosomal positions that exceeds the required depth (d) in all targeted loci. The proportion is highly correlated with PR score calculated using hotspots (R2 = 0.9694). The Journal of Molecular Diagnostics 2017 19, 651-658DOI: (10.1016/j.jmoldx.2017.06.001) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions