SCIENCE CHINA Life Sciences, Volume 62, Issue 1: 1-7(2019) https://doi.org/10.1007/s11427-018-9402-9

Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems

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  • ReceivedJul 18, 2018
  • AcceptedAug 19, 2018
  • PublishedNov 13, 2018


The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM (available at http://www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing (NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers, cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.

Funded by

the National Key Research and Development Program of China(2017YFD0102002)

the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences

and the National Natural Science Foundation of China(31401363)


We are extremely grateful to Ruiqiang Li from Novogene Bioinformatics Institute for critical reading of the manuscript. We thank Zheng Ruan and his team from Novogene Co., Ltd for NGS technical service. We also thank Yangwen Qian from Hangzhou Biogle Co., Ltd for rice transformation. This work was supported by the National Key Research and Development Program of China (2017YFD0102002), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and the National Natural Science Foundation of China (31401363).

Interest statement

The authors filed a patent application (Chinese patent application number 201710504178.3) based on the results reported in this paper.



Table S1 The primers developed for the PCR-based library construction kits

Table S2 Distribution of primer combinations in a 96-hole plate

Table S3 An example of sequence results in human cells

Table S4 An example of genotype results in human cells

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Schematic illustration of the workflow of Hi-TOM. A, The samples are amplified using site-specific primers (target-specific PCR). B, The products of the first-round PCR are used as templates for the second-round PCR (barcoding PCR) in the 96-hole plate kit. By barcoding PCR, the products of each sample are barcoded. C, All products of the second-round PCR are pooled in equimolar amounts in a single tube and sent for NGS. D, Hi-TOM analyses the data sample-by-sample and exports the results in Excel format.

  • Figure 2

    The home page of Hi-TOM and result of example. A, The home page of Hi-TOM. Job title stands for working directory; upload the NGS reads stands for uploading the forward reads (named_1.fq.gz) and reverse reads (named_2.fq.gz). B, An example of Hi-TOM sequence results. The results are summarised in the table. The results are populated in nine columns as follows: sample code and mutant name; read number; ratio; left variation type; left variation; right variation type; right variation; left read sequence; right read sequence. In the variation type volume, I, D and S indicate the insertion, deletion and SNP, respectively. For example, 3D represents the deletion of three bases and 1I represents insertion of one base. In the reads sequence column, the location of the mutation is indicated in lowercase. C, An example of Hi-TOM genotype results. AA stands for wild-type genotype; Aa stands for heterozygous genotype; aa stands for homozygous mutant genotype; * stands for in-frame mutation; # stands for SNP; - stands for missing data.

  • Figure 3

    The Sanger sequencing chromatograms and different mutation types tracked by Hi-TOM. H06 represents homozygous mutation; G06 indicates heterozygous mutation; F01 shows chimeric mutation with three variation types; G04 displays chimeric mutation with four variation types.

  • Figure 4

    The amplified fragments and Hi-TOM result of hexaploid wheat (Triticum aestivum, AABBDD). A, The amplified fragments of A, B and D genomes. The red letters indicate the target sites; the green letters represent sequence variations to distinguish between A genome and B genome; the blue letters show sequence variations to distinguish between A genome and D genome. B, The Hi-TOM result of hexaploid wheat. The mutation variations were displayed in the left variation type while the genomes were shown in the right variation type. WT indicates the A-genome, 8I exhibits the B-genome, and 6I represents the D-genome.

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