MOTIVATION: Low-frequency DNA mutations are often confounded with technical artifacts from sample preparation and sequencing. With unique molecular identifiers (UMIs), most of the sequencing errors can be corrected. However, errors before UMI tagging, such as DNA polymerase errors during end repair and the first PCR cycle, cannot be corrected with single-strand UMIs and impose fundamental limits to UMI-based variant calling. RESULTS: We developed smCounter2, a UMI-based variant caller for targeted sequencing data and an upgrade from the current version of smCounter. Compared to smCounter, smCounter2 features lower detection limit that decreases from 1 to 0.5%, better overall accuracy (particularly in non-coding regions), a consistent threshold that can be applied to both deep and shallow sequencing runs, and easier use via a Docker image and code for read pre-processing. We benchmarked smCounter2 against several state-of-the-art UMI-based variant calling methods using multiple datasets and demonstrated smCounter2's superior performance in detecting somatic variants. At the core of smCounter2 is a statistical test to determine whether the allele frequency of the putative variant is significantly above the background error rate, which was carefully modeled using an independent dataset. The improved accuracy in non-coding regions was mainly achieved using novel repetitive region filters that were specifically designed for UMI data. AVAILABILITY AND IMPLEMENTATION: The entire pipeline is available at https://github.com/qiaseq/qiaseq-dna under MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Next-generation sequencing (NGS) is rapidly becoming common practice in clinical diagnostics and cancer research. In addition to the detection of single nucleotide variants (SNVs), information on copy number variants (CNVs) is of great interest. Several algorithms exist to detect CNVs by analyzing whole genome sequencing data or data from samples enriched by hybridization-capture. PCR-enriched amplicon-sequencing data have special characteristics that have been taken into account by only one publicly available algorithm so far. RESULTS: We describe a new algorithm named quandico to detect copy number differences based on NGS data generated following PCR-enrichment. A weighted t-test statistic was applied to calculate probabilities (p-values) of copy number changes. We assessed the performance of the method using sequencing reads generated from reference DNA with known CNVs, and we were able to detect these variants with 98.6% sensitivity and 98.5% specificity which is significantly better than another recently described method for amplicon sequencing. The source code (R-package) of quandico is licensed under the GPLv3 and it is available at https://github.com/reineckef/quandico . CONCLUSION: We demonstrated that our new algorithm is suitable to call copy number changes using data from PCR-enriched samples with high sensitivity and specificity even for single copy differences.