BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment

Abstract

Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome.

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE

“…The data can be analysed by the CRISPR Genome Analyzer platform (CRISPR-GA), currently a popular bio-informatics tool that provides information on size and location of indels and on the efficiency of NHEJ and HDR events. A recently developed, easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data allows detection of indel mutations and other precise genome editing events and the rapid calculation of the corresponding mutagenesis efficiencies (Boel et al, 2016). This progress in off-target detection will help ensure that the CRISPR Cas or similar gene editing technologies work in a safe and accurate manner.…”

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models

“…(Center) Independent of the choice of targeted nuclease, HMA is used for qualitative genotyping of the F0 mosaic GEXM. If genome editing can be detected, qualitative genotyping is performed by targeted deep next‐generation sequencing and BATCH‐GE analysis (Boel et al ., )…”

“…(Center) Independent of the choice of targeted nuclease, HMA is used for qualitative genotyping of the F0 mosaic GEXM. If genome editing can be detected, qualitative genotyping is performed by targeted deep next-generation sequencing and BATCH-GE analysis (Boel et al, 2016) 2.2 | TALEN-mediated generation of GEXM Although the CRISPR/Cas9 technique is preferred because of the high speed, efficiency, and simplicity, TALEN technology would be the technique of choice for targeting certain sites recalcitrant to CRISPR/Cas9 editing ( Figure 1b). Since with the classical CRISPR/Cas9 system, sgRNA targeting sites need to be followed by the protospacer adjacent motif (PAM) NGG-3 0 , it may be impossible to find a sgRNA with sufficient predicted in vivo effectiveness for the desired target, especially in AT-rich sequences.…”

“…We employ HMA as a rapid and cost effective qualitative method for initial genome modification assessment (Figure 1c). If positive, subsequent batch analysis of next-generation targeted deep sequencing data (BATCH-GE) provides a rapid and very cost-effective platform for quantitative evaluation of CRISPR/Cas9 based experiments (Boel et al, 2016). This method allows for both accurate quantification of genome editing efficiencies and provides specific frequencies of each sequence variant thus fully resolving the complexity of the F0 mosaic GEXM (Naert et al, 2016).…”

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

“…Alternative computational tools to CRISPResso exist to evaluate genome-editing outcomes from deep-sequencing data 57–61 ; however, these tools offer limited analysis functionality for pooled amplicon sequencing or WGS data as compared with the CRISPResso suite. CrispRVariants is another tool that offers functionality to analyze deep-sequencing data by quantifying mosaicism and allele-specific gene editing, as well as multisequence alignment views 60 .…”