Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid Meenakshi Mehrotra, Rajesh.

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Presentation transcript:

Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid Meenakshi Mehrotra, Rajesh R. Singh, Wei Chen, Richard S.P. Huang, Alaa A. Almohammedsalim, Bedia A. Barkoh, Crystal M. Simien, Marcos Hernandez, Carmen Behrens, Keyur P. Patel, Mark J. Routbort, Russell R. Broaddus, L. Jeffrey Medeiros, Ignacio I. Wistuba, Scott Kopetz, Rajyalakshmi Luthra The Journal of Molecular Diagnostics Volume 19, Issue 4, Pages 514-524 (July 2017) DOI: 10.1016/j.jmoldx.2017.03.003 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Quantitative and qualitative analysis of cfDNA. A: cfDNA yield from plasma samples separated and extracted by Qiamp Manual extraction method using Qiagen circulating nucleic acid kit at different time intervals [2 (red dots), 4 (blue dots), 16 (black dots), and >24 (green dots) hours] from peripheral blood collected in cell-stabilizing Streck tubes or in standard K3-EDTA tubes. B: Correlation between Streck and K3-EDTA tubes for cfDNA yield. C: Box plot showing integrity index (ratio of Alu247/Alu115) of cfDNA extracted from Streck and K3-EDTA collection tubes calculated using Alu quantitative PCR. ∗∗P < 0.01, ∗∗∗∗P < 0.0001. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Scalability of mutation detection by the Ion Proton platform. A: Comparison of Ion PGM (4 samples per run) and Ion Proton (31 samples per run) platforms for coverage and variant allele frequencies in cfDNA extracted from the same matched plasma samples separated from peripheral blood collected in Streck tubes. B: Correlation between Ion PGM and Ion Proton platforms for mutant allelic frequency in cfDNA. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Sensitivity of Ion Proton and MiSeq platforms for mutation detection in cfDNA samples. A serial dilution study of a patient cfDNA sample positive for KRAS p.G12D (36%) and SMAD4 p.R361C (45.09%) mutations was used to compare the sensitivity of the two platforms. The positive cfDNA was diluted with a cfDNA sample negative for these mutations to obtain 50%, 25%, 12.5%, 6.25%, 3.15%, 1.5%, or 0.75% positive (mutated) DNA in wild-type DNA. The results for the Ion PGM (12 samples per run; A), Ion PGM (4 samples per run; B), Ion Proton (31 samples per run; C), and MiSeq (12 samples per run; D) platforms for sensitivity studies are shown. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Comparison of Ion Proton and MiSeq platforms for cfDNA sequencing. Fifty-two mutations detected in 31 cfDNA plasma samples, with advanced cancer of various types detected by both the platforms, were compared. A: Percentage mutation rates for the CTNNB1, FBXW7, TP53, KRAS, SMAD4, PIK3CA, APC, EGFR, CDKN2A, JAK3, IDH1, and ALK genes in sequenced tumor DNA and cfDNA (plasma) samples of 31 patients. B: The symbol and line plot compares mutant allelic frequencies of Ion Proton and MiSeq for variants detected in cfDNA samples. Each dot represents a single mutant, whereas the lines connect the same mutant on the other platform. C: Venn diagram showing concordance for mutants detected in cfDNA on the Ion Proton and MiSeq platforms. D: The correlation between Ion Proton and MiSeq for mutant allelic frequency in sequenced cfDNA samples. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Custom bioinformatics pipeline for analysis of MiSeq sequencing data for cfDNA. QC, quality control. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Qualitative analysis of cfDNA extracted from plasma separated from peripheral blood drawn into Streck (S) or EDTA (E) tubes. A: Electropherogram of cfDNA samples. P1, P2, P3, P4, and P5 represent five different cfDNA samples run on Agilent Bioanalyzer. B–G: Qualitative analysis comparing results for plasma cfDNA samples separated at different time intervals from peripheral blood collected in Streck or EDTA tubes: 2 (B and C), 4 (D and E), and 16 (F and G) hours represented by fluorescent unit (FU). Size of cfDNA (120 bp), lower marker (LM), and upper marker (UM) are represented by arrows. NTC, nontemplate control. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 Quality control metrics for cfDNA libraries loaded on Ion PGM 318 version 2 chip (four samples per run) at different loading concentrations (20, 12, 10, and 5 pmol/L). A: Total reads obtained on Ion PGM after sequencing for each concentration. B: Average polyclonal frequency obtained after loading different concentration on Ion PGM. C: Mapped reads and mean depths obtained for cfDNA samples sequenced at different library loading concentrations. D: Coverage. E: Allelic frequency of variants sequenced at different library loading concentrations. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Downstream sequencing analysis comparison for cfDNA extracted from different collection tubes sequenced on Ion PGM. A: Comparison of variant allele frequencies in cfDNA sequenced on an Ion PGM. B: Correlation between variant allele frequencies (AFs) for cfDNA. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S4 Scalability of cfDNA sequencing on the Ion Proton platform: Comparison of sequencing quality metrics using different semiconductor sequencing platforms (Ion PGM and Ion Proton). A: Comparison of number of samples and total reads obtained per run on the Ion PGM (four samples per run) and Ion Proton platforms. The number of samples/run is shown. B: Comparison of polyclonal frequencies obtained by sequencing the same cfDNA libraries on Ion Proton and Ion PGM platforms. C: Mapped reads and mean depths for sequenced cfDNA samples extracted from plasma Streck tubes on Ion PGM (4 samples/run) and Ion Proton (31 samples/run). The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S5 Integrative genomic view of new clones detected in plasma cfDNA sequencing but absent in tissue by Ion Proton and MiSeq KRASp.Q61H (A) and EZH2p.Y646F (B), new mutations in cfDNA extracted from plasma and detected by Ion Proton and MiSeq platforms studied. Each view compares results with those in tumor tissue. The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S6 Summary of MiSeq data analysis of common variants selected from the in-house and the Swift Bioinformatics pipelines. Bar graphs show variant allele frequencies (A), variant coverage (B), and coverage after application of filter 1 (varfreq ≥0.01, varcov ≥10, coverage ≥250) and filter 2 (ie, after removal of intron, synonymous, untranslated region, upstream, downstream, and intronic modifiers, and SPLICE_SITE_DONOR+INTRON) (C). D: Venn diagram showing number of single-nucleotide variants (SNVs) and insertions/deletions (indels) among common variants. n = 352 (D). The Journal of Molecular Diagnostics 2017 19, 514-524DOI: (10.1016/j.jmoldx.2017.03.003) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions