Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data

Abstract

Motivation: Because of its low cost, amplicon sequencing, also known as ultra-deep targeted sequencing, is now becoming widely used in oncology for detection of actionable mutations, i.e. mutations influencing cell sensitivity to targeted therapies. Amplicon sequencing is based on the polymerase chain reaction amplification of the regions of interest, a process that considerably distorts the information on copy numbers initially present in the tumor DNA. Therefore, additional experiments such as single nucleotide polymorphism (SNP) or comparative genomic hybridization (CGH) arrays often complement amplicon sequencing in clinics to identify copy number status of genes whose amplification or deletion has direct consequences on the efficacy of a particular cancer treatment. So far, there has been no proven method to extract the information on gene copy number aberrations based solely on amplicon sequencing.Results: Here we present ONCOCNV, a method that includes a multifactor normalization and annotation technique enabling the detection of large copy number changes from amplicon sequencing data. We validated our approach on high and low amplicon density datasets and demonstrated that ONCOCNV can achieve a precision comparable with that of array CGH techniques in detecting copy number aberrations. Thus, ONCOCNV applied on amplicon sequencing data would make the use of additional array CGH or SNP array experiments unnecessary.Availability and implementation: http://oncocnv.curie.fr/Contact: valentina.boeva@curie.frSupplementary information: Supplementary data are available at Bioinformatics online.

Genomic landscapes of breast fibroepithelial tumors

“…Copy number estimates for each of the genes in the targeted sequencing study were obtained using OncoCNV 22 . The algorithm uses a pool of reference samples to construct a baseline that can be used to infer probable somatic CNAs.…”

Study of chromatin remodeling genes implicates SMARCA4 as a putative player in oncogenesis in neuroblastoma

“…The FW and RV normalized data were then merged to reconstruct copy number profiles. The copy number profile obtained from TSCA data using our in‐house developed pipeline was also confirmed by another technique ONCOCNV, a method that includes a multifactor normalization with respect to library size, CG‐content and target length to detection of large copy number changes from amplicon deep sequencing data …”

Altered molecular profile in thyroid cancers from patients affected by the Three Mile Island nuclear accident

“…In addition to gene fusions, somatic copy number alterations (SCNAs) may play an important role in the oncogenesis of sporadic and radiation‐induced thyroid cancer . To identify copy number alterations in our amplicon sequencing data, we used the ONCOCNV algorithm, which achieves a degree of precision similar to more traditional comparative genomic hybridization arrays . Using this approach, we identified SCNA driver mutations in two tumors from the at‐risk group (13%) and one tumor from the control group (3%) in which we identified no other oncogenic driver mutations.…”

“…Finally, all called indels were filtered based on score, frequency, and strand bias. Somatic copy number variants were called using the ONCOCNV algorithm for amplicon‐based sequencing data …”