KAPA hgdna QUANTIFICATION AND QC KIT:

Transcription

1 Poster Note As presented at AGBT 2015, Marco Island, FL KAPA hgdna QUANTIFICATION AND QC KIT: The KAPA Human Genomic DNA Quantification and QC Kit Enables Prediction of Sequencing Performance through User-Defined Metrics Authors Francine de Abreu 1, Jason D. Peterson 1, Rachel W. Kasinskas 2, Brian Komorous 2, Gregory J. Tsongalis 1 1 Dartmouth-Hitchcock Medical Center, 1 Medical Center Dr, Lebanon, NH 2 Kapa Biosystems, 200 Ballardvale St, Suite 250, Wilmington, MA

2 INTRODUCTION Applications that utilize next-generation sequencing to identify clinically actionable genomic alterations require fast turnaround time, minimized cost, and reproducible results. The challenge with clinical oncology samples (e.g., FFPE) is processing them in a standard library preparation pipeline with a predictable success rate. Low-quality, FFPE samples are often limiting in quantity and contain DNA modifications resulting in a lower conversion of input DNA to library molecules; the impact on sequencing is lower library diversity, higher duplication rates, and lower sequence coverage. In this study, genomic DNA from 72 FFPE tumor samples were screened for both quantity and quality using the KAPA Human Genomic DNA Quantification and QC Kit (KAPA hgdna QQC), which amplifies targets of 41 bp, 129 bp, and 305 bp within a conserved single-copy locus in the human genome. Normalization of the concentrations of the longer amplicons to the 41 bp amplicon is calculated for quality, termed Q-ratio. Samples consisting of high-quality DNA will result in Q-ratios close to 1, while damaged DNA will result in Q-ratios significantly less than 1. For comparison, quality was also assessed for a subset of samples with the Illumina FFPE QC Kit. Amplicon libraries were prepared using both the Ion AmpliSeq Cancer Hotspot Panel v2 and the Illumina TruSeq Amplicon Cancer Panel workflows, and sequenced on the Ion Torrent PGM and Illumina MiSeq platforms, respectively.

3 FIGURE 1 Principle of the KAPA Human Genomic DNA Quantification and QC Kit 1A High-quality hgdna bp bp bp Q 129 bp Q 41 bp = 1 Q 305 bp Q 41 bp = 1 Damaged hgdna hgdna bp bp bp Q 129 bp Q 41 bp < 1 Q 305 bp Q 41 bp < 1 1B 41 bp 129 bp 305 bp 84.5 C 72.5 C 86.3 C A A single set of DNA Standards is used to generate up to three standard curves, using three different primer pairs that amplify targets of 41 bp, 129 bp or 305 bp within a conserved, single-copy human locus. The 41 bp assay is used for absolute quantification of DNA samples. For an assessment of DNA quality, standard curves are generated and samples assayed with the 129 bp and/or 305 bp primer premix(es). Since poor DNA quality has a greater impact on the amplification of longer targets, the relative quality of a DNA samples can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained from the 41 bp assay. This normalization generates a Q-ratio (with a value between 0 and 1) that can be used as a relative measure of DNA quality. Typically, low-quality samples will give the following Q-ratios: Q(129/41)<1 and Q(305/41)<<1. B Each of the three amplicons generated using this kit (41 bp, 129 bp, and 305 bp) has a specific melting temperature, 72.5 C, 84.5 C, and 86.3 C, respectively. It is recommended that a melt curve analysis be performed to confirm that specific product in each reaction. Melt curve analysis may also be helpful is identifying cross-contamination, misidentification, or accidental mix-up of Primer Premixes. FIGURE 2 Experimental Design Total of Samples N=72 Sequenced N=36 QNS N=36 PGM N=28+8 MiSeq N=8 Genomic DNA from 72 formalin-fixed paraffin-embedded (FFPE) tissue samples was assayed with the KAPA hgdna QQC Kit. Based upon previous library preparation results, samples were divided into two categories: 36 Quality Not Sufficient (QNS) and 36 sequenced samples. Of the 36 sequenced samples, 28 were only sequenced on the Ion Torrent PGM, and eight were sequenced on both the Ion Torrent PGM and Illumina MiSeq platforms.

4 ESTABLISH SAMPLE QUALITY CONTROL MINIMUMS The Translational Research Laboratory at Dartmouth-Hitchcock Medical Center (DHMC) utilizes internal quality control assays to assess workflow performance at specific steps throughout sample processing, including after DNA extraction, library preparation, emulsions PCR and enrichment, and data analysis. Most samples tend to fail after library preparation, which entails two days of sample processing. Those samples that did not pass library QC were identified as Quality Not Sufficent (QNS) and sequencing was not performed. 2 FIGURE 3 Q-ratios for sequence and QNS d samples Q129 bp / Q41 bp ratio Q305 bp / Q41 bp ratio Sequenced Samples Q129 bp / Q41 bp ratio Q305 bp / Q41 bp ratio Sequenced Samples (High-quality DNA) Q129bp/Q41bp: Q305bp/Q41bp: DHMC QC Ratio Q129/Q41 > 0.5 Q305/Q41 > 0.2 QNS d Samples < 0.5 < QNS d Samples (Low-quality DNA) Q129bp/Q41bp: <0.5 Q305bp/Q41bp: <0.2 Implementation of the KAPA hgdna Quantification and QC Kit after DNA extraction, but prior to library preparation enabled the team to establish an additional internal quality control metric that could be utilized to assess sample quality earlier in the process. DHMC QC ratios of Q129/41 > 0.5 and Q305/41 > 0.2 were empirically determined. FIGURE 4 Sequencing results for samples that passed the DHMC QC Ratio qpcr Quantification Mean Target Coverage % Reads on Target % Uniformity of Coverage Total Reads Min Max Average Median For those samples that passed the DHMC QC Ratios (N=36), minimum PGM sequencing performance was defined: mean target coverage >1000, percent reads on target >80%, and percent uniformity of coverage >80%. Samples that did not pass the DHMC QC Ratio were considered poor quality, and not processed further.

5 DETERMINE Q-RATIOS FOR SEQUENCING APPLICATIONS In order to verify the utility and accuracy of the DHMC QC Ratio as a predictor for sequencing performance, libraries were prepared for a subset of samples and sequenced both on the Ion Torrent Personal Genome Machine (PGM) and Illumina MiSeq. Eight libraries were assayed with both the KAPA hgdna QQC Kit and the Illumina FFPE QC Kit. Two libraries were prepared from each sample. One library was prepared with the Ion AmpliSeq Library Kit 2.0 with the Cancer HotSpot Panel v2, a PCR-based, targeted amplicon sequencing workflow that utilizes 10 ng of FFPE genomic DNA as input. The second library was prepared with the Illumina TruSeq Amplicon Custom Cancer Library Prep Kit, a hybridization, extension, and ligation-based targeted amplicon sequencing workflow that requires a minimum of 250 ng of FFPE genomic DNA. Each library was sequenced on the respective sequencing platform, i.e. Ion Torrent PGM or Illumina MiSeq. FIGURE 5 Sequencing application effects the utility of the DHMA QC Ratio KAPA hgdna QQC Kit FFPE QC Kit PGM Metrics MiSeq Metrics Q129/Q41 Q305/Q41 ΔCq Mean Target Coverage Percent Reads on Target Percent Uniformity of Coverage Number of Variants (250 ng) Number of Variants (100 ng) Sample Sample Sample Sample Sample Sample Sample Sample Of the eight samples that were QC d by both methods, three samples each passed (samples 2, 3, 8) and failed (samples 1, 5, 6) all metrics. The three samples that passed both QC methods produced expected sequencing results, i.e. passed samples achieved a mean target coverage >1000, percent reads on target >80%, and percent uniformity of coverage >80%, whereas the failed samples did not (data not shown). The remaining two samples (as outlined above) both passed the KAPA Q129/Q41 ratio, but failed the KAPA Q305/41 ratio; however, only Sample 7 passed the Illumina FFPE QC metric. Of these two samples, both passed PGM sequence metric minimums, but neither resulted in expected variant calls with MiSeq sequencing. To understand the relationship between pass/fail metrics and sequencing results, the required read length for each library preparation workflow should be understood. For PGM sequencing, the Ion AmpliSeq Library Prep workflow requires an average insert size of bp. For MiSeq sequencing, the TruSeq Ampicon Custom Cancer Panel Library Prep workflow requires an average insert size of 300 bp. Based upon the sequencing results, Samples 4 and 7 resulted in high-quality PGM data, even though neither sample passed the Q(305/41) ratio. This indicates that for amplicon-based workflows that utilize a library fragment size <150 bp, the Q(129/41) ratio is most predictive of performance. On the other hand, for sequencing applications that require insert sizes >150 bp, both the Q(129/41) and Q(305/41) ratios are required to predict sequence performance. In this data set, both Samples 4 and 7 failed the Q(305/41) ratio, and resulted in unacceptable sequencing results on the MiSeq platform. Moreover, results with the FFPE QC Kit were inconsistent, further supporting that a more precise determination of pass/fail metrics can be achieved with the KAPA hgdna QQC Kit.

6 OBTAIN REPRODUCIBLE RESULTS To assess the reproducibility of the KAPA hgdna Quantification and QC Kit, genomic DNA from five samples were assayed for both Q129/Q41 and Q305/Q41 ratios in separate experiments on two different days. FIGURE 6 Comparison between Q-ratios measured through independent experiments Q(129/41) Q(305/41) In all five samples, consistent results were obtained for both the Q(129/41) and Q(305/41) metrics. Q-Ratio A B C D E Sample

7 CONCLUSIONS The KAPA Human Genomic DNA Quantification and QC Kit does not assign a pass/fail value to samples, but allows user-defined values based upon the application. Furthermore, the sequencing application and platform has an impact on how the QC metric should be defined. For amplicon sequencing, the following metrics were used to identify the inflection point for sequencing performance of FFPE samples: Target read length > 150 bp: Q-ratio of Q(129/41) > 0.5 and Q(305/41) > 0.2 Target read length < 150 bp: Q(129/41) > 0.5

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