Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 51 (2018)

Trials of mutation detection programs to detect structural variations
induced by heavy-ion beams in rice
R. Morita,∗1 H. Ichida,∗1 Y. Shirakawa,∗1 and T. Abe∗1
We screened rice mutants generated by heavy-ionbeam irradiation and identified mutations using wholegenome sequencing (WGS). A heavy-ion beam induces
both small mutations (single-nucleotide variants andsmall insertions/deletions) and structural variations(SVs) such as large deletions, inversions, and translocations.1) The detection of SVs using short-read WGS
data is a challenging task compared with the detection of small mutations.2) We used the Pindel3) software to detect SVs. Pindel can detect break points oflarge deletions (< 10 kb) and small-size insertions (1–20 bp).3) However, the heavy-ion beam can generatedeletions with lengths of over 10 kb.1) Therefore, besides Pindel, another program is needed to detect theSVs induced by the heavy-ion-beam irradiation.
In the present study, we tested two programs,Delly4) and Manta,5) for detecting the SVs inducedby heavy-ion irradiation. We used a high-performancebioinformatics pipeline6) incorporated with Delly andManta. The candidate mutations were visually confirmed by using Integrated Genomics Viewer (IGV).
We analyzed WGS data of 11 rice mutants inducedby carbon ion irradiations (12 C6+ , 50–175 Gy, LET:
30 keVµm−1 ). In the present study, we ignored
the candidates of heterozygous mutations detected by
Delly and Manta because all heterozygous mutations
were false-positives in our initial experiment (Data notshown).
In the 11 mutants, there were 10 and 19 homozygous
candidates detected by Delly and Manta, respectively.
Of the 10 and 19 candidates detected by Delly andManta, 8 and 9 were positive mutations, respectively.
These data suggest that both programs detected positive mutations induced by carbon ions. Of the 8 and9 positive mutations detected by Delly and Manta, 4and 2 were also detected by Pindel. In other words,
both 4 mutations detected by Delly and 7 mutations
detected by Manta were not detected by Pindel. In
total, 8 independent mutations that were not detectedby Pindel were identified (Table 1), i.e. one mutation(No. 1) was detected by Delly only, 4 mutations (No. 2,5, 7, and 8) were detected by Manta only, and 3 mutations (No. 3, 4, and 6) were detected by both programs.
Using IGV, we determined the types of each mutation.
One mutation (No. 1) was determined to be a deletion (Table 1). The remaining mutations (No. 2, 3,4, 5, 6, 7, and 8) were estimated to be translocations(Table 1). We tried to design primer pairs around thebreakpoints of each mutation to confirm the existenceof mutation by PCR analysis. Around the mutation∗1RIKEN Nishina Center- 237 -Fig. 1. PCR confirmation of 7 mutations. W indicates theresult of using wild-type DNA as the template, and mindicates the result of using mutant DNA as the template. The PCR pruducts are analyzed using MultiNA
MCE-202 (Shimadzu). DNA size markers are shown on
the left side.
Table 1. Structural variations detected by Delly andManta.
ND, not determined by

No. 4, a primer pair specific to the wild-type allele
could not be designed. When we performed PCR withthe 7 primer pairs using wild-type genomic DNA asa template, each band was detected (Fig. 1, lane W).
However, no band wasdetected when we used mutant genomic DNA as atemplate (Fig. 1, lane m), indicating that the wildtype alleles do not exist in these mutants. Our present
study indicated that both Delly and Manta are usefulprograms to detect SVs in rice mutants induced byheavy-ion irradiation.
References
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3) K. Ye et al., Bioinformatics 25, 2865 (2009).
4) T. Rausch et al., Bioinformatics 28, 1333 (2012).
5) X. Chen et al., Bioinformatics 32, 1220 (2016).
6) H. Ichida et al., RIKEN Accel. Prog. Rep. 49, 254(2016).