Assessing Copy Number Alterations in Targeted, Amplicon-Based Next-Generation Sequencing Data

Abstract

Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>j2j copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage. (J Mol Diagn 2015, 17: 53e63; http://dx

Gene Copy Number Estimation from Targeted Next-Generation Sequencing of Prostate Cancer Biopsies: Analytic Validation and Clinical Qualification

“…1B); however, the CNA estimation confidence score (IQR value) was improved when using pooled germline samples (paired t test P ¼ 0.05). This was in line with other reports (14) that indicated that using pooled germline sequencing data as reference offered a robust method to identify gene CNAs.…”

“…Accurately assessing the depth of coverage of genes can be biased by high GC content and repetitive regions (11,14), so we first evaluated CNA reproducibility on a per-gene basis between technical replicates. Thirteen samples were sequenced in duplicate to assess technical variation; 12 of these duplicate datasets included repeated library preparation from the same DNA extraction, while one involved resequencing of the same library twice.…”

Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies

“…These findings are consistent with those reported by other investigators who have also recently demonstrated reliable detection of focal CNV loss or gain with the use of amplification-based sequencing data. 27,28 For annotation, we elected to limit the classification of variants as either pathogenic or variant of unknown significance. We used a strategy of treating oncogenes and tumor suppressor genes in two different ways in assigning pathogenicity.…”

Identification of TargetableERBB2Aberrations in Head and Neck Squamous Cell Carcinoma

“…the difference in the number of forward vs. reverse reads containing the variant allele) >5-fold were removed from the variant set, as previously described 18 . Copy number alterations were identified clinically relevant at >|2| copies, as previously described 18,20 , using normalized, GC content corrected, total read counts per amplicon from each sample divided by those from a composite “normal” sample consisting of multiple single and pooled normal male DNA samples. Gene-level (e.g.…”

“…Gene-level (e.g. HER2 ) copy number estimates were determined by taking the coverage-weighted mean of the per-probe ratios, with expected error determined by the probe-to-probe variance, as previously described 20 .…”