Josh D. Haimes1, Namitha Manoj1, Abel Licon1, Joshua A. Stahl1, Brian A. Kudlow1, Maria Cueller2, Milhan Telatar2
1ArcherDX, Inc., Boulder, CO; 2Molecular Diagnostics Lab, City of Hope Medical Center, Los Angeles, CA
Copy number variation (CNV) results from oncogene amplification or tumor suppressor gene deletion and is a common mode of gene deregulation in cancer (1). Several techniques have been employed to determine copy number, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR); however, none of these methods are amenable to high-throughput, directed CNV detection. We developed a directed next-generation sequencing (NGS)-based method to rapidly and quantitatively measure the copy number of tens and potentially hundreds of genes simultaneously. This complete workflow, found in the Archer™ Universal DNA Kit, is powered by Anchored Multiplex PCR (AMP™) chemistry and processes dozens of samples in about 6 hours. By ligating a molecular barcode to randomly fragmented input DNA and then using AMP to simultaneously enrich for several regions of each target gene in test samples by counting unique molecular barcodes associated with each target region.
We validated our methodology with a 25-gene panel on a subset of NCI-60 cell lines by comparing our copy number measurements to those determined by both aCGH and qPCR. Results from both orthogonal methods strongly correlated with data from our NGS-based method. We multiplexed hundreds of samples on a single MiSeq® run and detected CNVs, both amplifications and deletions, of 2X magnitudes (and often lower) at extremely high confidence, indicating that this panel is amenable to highly multiplexed screens of potentially hundreds of samples. Furthermore, we demonstrate that our NGS-based CNV detection workflow and analysis is compatible with DNA extracted from formalin-fixed, paraffin embedded (FFPE) samples, suggesting that this system could be adapted for use in clinical applications.
Published in conjunction with City of Hope™
A. Genomic regions interrogated in the 25-gene oncogene/tumor suppressor panel. Target genes are listed in red, and control genes are listed in blue.
B. Overview of CNV detection strategy. Target gene regions are uniquely tagged with molecular barcodes (MBCs) and enriched for NGS analysis by Anchored Multiplex PCR (AMP). The number of unique MBCs associated with each target region is used to deduce copy number for each target region relative to a control sample.
C. Archer Universal DNA Workflow. Library preparation is carried out using Archer's fully lyophilized reagents. Input DNA, as little as 10 ng, is enzymatically sheared, tagged and amplified into sequencer-ready indexed libraries, allowing hundreds of samples to be multiplexed. Total library preparation time is about 6 hours for dozens of samples.
D. Overview of Archer Analysis for CNV detection. CNV analysis is fully automated, permitting sensitive detection of CNVs without a need for bioinformatics training.
A. Archer NGS-based CNV assay correlates closely with published aCGH data. Copy number of target genes was measured in MCF-7 and HT-29 cell lines with the Archer CNV assay, and copy number calls were compared to published aCGH data (2). The correlation between copy number calls is shown in the graphs.
B. Archer CNV assay correlates closely with qPCR copy number measurements. Copy number of target genes was measured in MCF-7 and HT-29 cell lines with the Archer CNV assay. A single qPCR probe for each target was then used to measure copy number in the same genomic DNA samples. The correlation between copy number calls is shown in the graphs.
C. High concordance between Archer NGS assay and published aCGH data over complete panel. Table shows copy number calls across complete panel in MCF-7 cells.
|Gene||Copy Number (aCGH)||Copy Number (Archer)|
A. Multiplexing options available with Archer Universal DNA Kit. Archer NGS assays facilitate highly multiplexed CNV measurements with 144 P5 adapter indexes and 8 P7 adapter indexes for a total of 1,152 dual-index combinations.
|P5 1||P5 2||P5 3||P5 4||...||P5 144|
B. Highly sensitive CNV calls are achievable even at minimal sequencing depth. MCF-7 CNV libraries were sub-sampled to sequencing depths equivalent to indicated multiplexing (assuming 15M productive reads per MiSeq run). P-values for significant CNV calls are plotted against the read depth. Even at sequencing depths equivalent to running 1500 samples on a single MiSeq run, modest CNVs (e.g. FGFR1) are called with p < 0.01.
A. Overview of qPCR input QC assay. Probe-based measurement of quantity of amplifiable DNA present in an FFPE input. Amplicon size is 66 bp.
B. qPCR QC assay predicts ability to call CNVs. Receiver operating characteristic (ROC) plots demonstrating predictive power of input QC assay in identifying samples in which a CNV of magnitude 3X(left) or 4X (right) could be called by the Archer CNV assay.The minimum magnitude of a CNV is a function of baseline noise, which increases as input quality decreases. Data is representative of 43 archived FFPE samples.
C. qPCR assay predicts magnitude of detectable CNV. The sensitivity of the Archer CNV assay, expressed as the minimum detectable CNV, for a given sample is linearly correlated with the Cq value of input QC qPCR. Poor quality samples result in higher input Cq values, and this is correlated with reduced CNV sensitivity. Total input quantity was equivalent for all samples.
A. Copy number report for four clinical FFPE samples. Copy number variants identified in each of four clinical samples are shown. Each bar represents a single probe, where the magnitude of the copy number (log 2) corresponds to the height of the bar and the significance is shown by color (z-score, scale below).
B. Description of the samples in A. Input quantity and expected CNVs for each sample in A.
|Sample||pPCR QC||Input (ng)||Input Type||Tissue||Tumor Cellularity||CNVs|
C. qPCR confirmation of detected CNVs. CNVs detected in the three samples were validated by qPCR with a single primer set for each of the indicated genes.
A. Archer assay detects copy number variants in patient samples. Forty-seven archived clinical FFPE samples were screened with the qPCR QC assay. Libraries were generated and sequenced for the 21 samples that passed the qPCR QC assay as well as 10 of the 16 samples that failed QC. Samples are ordered according to input quality determined by qPCR QC. QC pass threshold was determined by ROC analysis. Copy number is reported for all samples; those with greater than 2-fold copy number for control genes in samples that passed QC was 1.03 +/- 0.33 and for samples that failed QC 1.70 +/- 3.42, indicating that samples that failed QC have insufficient signal-to-noise ratio to support CNV calling.
B. qPCR confirmation of selected CNVs. Selected CNVs identified by Archer CNV assay were confirmed by qPCR. Copy number measurements tightly correlate with qPCR measurements for high quality samples (top) but not for samples that fail input QC (bottom).
For Research Use Only. MiSeq® is a registered trademark of Illumina, Inc. Archer™ and FusionPlex™ are trademarks of ArcherDX, Inc.
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