Motivation: PCR-based DNA enrichment followed by massively parallel sequencing is a straightforward and cost effective method to sequence genes up to high depth. The full potential of ampliconbased sequencing assays is currently not achieved as analysis methods do not take into account the source amplicons of the detected variants. Tracking the source amplicons has the potential to identify systematic biases, enhance variant calling and improve the designs of future assays. Results: We present Nimbus, a software suite for the analysis of amplicon-based sequencing data. Nimbus includes tools for data pre-processing, alignment, single nucleotide polymorphism (SNP), insertion and deletion calling, quality control and visualization. Nimbus can detect SNPs in its alignment seeds and reduces alignment issues by the usage of decoy amplicons. Tracking the amplicons throughout analysis allows easy and fast design optimization by amplicon performance comparison. It enables detection of probable false positive variants present in a single amplicon from real variants present in multiple amplicons and provides multiple sample visualization. Nimbus was tested using HaloPlex Exome datasets and outperforms other callers for low-frequency variants. The variants called by Nimbus were highly concordant between twin samples and SNP-arrays. The Nimbus suite provides an end-to-end solution for variant calling, design optimization and visualization of amplicon-derived next-generation sequencing datasets.,
Erasmus MC: University Medical Center Rotterdam

Brouwer, R., van den hout, M., Kockx, C., Brosens, E., Eussen, B., de Klein, A., … van IJcken, W. (2018). Nimbus: A design-driven analyses suite for amplicon-based NGS data. Bioinformatics, 34(16), 2732–2739. doi:10.1093/bioinformatics/bty145