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Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Abstract Background Even subtle changes in environmental factors can exert behavioral effects on creatures, which may alter interspecific interactions and eventually affect the ecosystem. However, how changes in environmental factors impact complex behaviors regulated by neural processes is largely unknown. The freshwater planarian Dugesia japonica, a free-living flatworm, displays distinct behavioral traits mediated by sensitive perception of environmental cues. Planarians are thus useful organisms for examining interactions between environmental changes and specific behaviors of animals. Results Here we found that feeding behavior was suppressed when the concentration of ions in the breeding water was low, while other behaviors were unaffected, resulting in differences in population size. Notably, the decline in feeding behavior was reversed in an ion-concentration-dependent manner soon after the planarians were moved to ion-containing water, which suggests that ions in environmental water rapidly promote feeding behavior in planarians. Moreover, the concentration of ions in the environmental water affected the feeding behavior by modulating the sensitivity of the response to foods. Finally, we found that calcium ions in the aquatic environment were required for the feeding behavior, and exposure to higher levels of calcium ions enhanced the feeding behavior, showing that there was a good correlation between the concentration of calcium ions and the responsiveness of planarians to foods. Conclusions Environmental calcium ions are indispensable for and potentiate the activity level of the feeding behavior of planarians. Our findings suggest that the ions in the aquatic environment profoundly impact the growth and survival of aquatic animals via modulating their neural activities and behaviors.