Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 49 (2016)

Development of a high-performance bioinformatics pipeline for riceexome sequencing
H. Ichida,∗1 Y. Shirakawa,∗1 R. Morita,∗1 Y. Hayashi,∗1 and T. Abe∗1
Heavy-ion beams cause DNA double strand breaks
in a cell along with the beam direction and induce mutations, including insertions, deletions, inversions, andbase substitutions in genome. Recent advances in massively parallel (aka “next generation”) DNA sequencing technology enabled us to perform a comprehensive
analysis of genomic mutations in base-pair resolution.
We have been establishing heavy ion beam-irradiatedrice progenies as a bioresource that are subjected toscreening experiments to meet scientiﬁc and agricultural needs. Although among crops, rice has a relatively small genome size (approximately 400 million
bases per haploid), most parts are ﬁlled with repetitive and junk elements and only 10% is predicted toencode proteins. We have developed a system to enrich rice exons, which are genomic regions transcribedand translated into proteins, and to determine their
nucleotide sequences. By this strategy, called exome
sequencing, it is possible to reduce sequencing costs
signiﬁcantly by eliminating meaningless sequences andfocusing on the regions encoding proteins.
In the present study, we have developed a highperformance bioinformatics pipeline for analyzing riceexome sequencing results, and we identify the most signiﬁcant mutations without the need for prior knowledge. The pipeline was implemented on Hokusai
GreatWave, which is a parallel computing platform operated by Advanced Center for Computing and Communication, RIKEN. In the pipeline, raw sequencing
reads (100–150 bp in length) are mapped to reference
Nipponbare sequences using Burrows-Wheeler Aligner(BWA) software1) , sorted, and realigned, and the datais stored in the standard BAM format. Programs with
3 distinct algorithms (GATK2) , Pindel3) , and Bedtools4) ) are used to identify the mutations. We implemented a ﬁlter to remove duplicated and unreliable
mutation candidates. The list of mutations were storedin tab-delimited text ﬁle and accessible from genericspreadsheet software. In parallel, the quality of raw
reads were checked by FastQC5) program and the results were stored as an HTML-formatted report for review. The entire process was executed automaticallyand in parallel through a batch job controlling system.
The deployment of an in-house bioinformatics pipeline
enabled reliable comparison of results from diﬀerent
experiments (batches), since the pipeline is controlled
by a versioning system; therefore, it is possible to reproduce exactly the same program and database versions any time.
To test the validity of variant calling and the fol∗1RIKEN Nishina Center- 254 -lowing ﬁltration, we analyzed the exome sequencingresults from 8 individual rice mutants obtained via
carbon- and neon-ion irradiations. As the result, there
were 62,024 mutation candidates in GATK and Pindel outputs, but most of those mutations are common among the mutants and are likely to be intracultivar polymorphisms between our parental Nipponbare line and the reference sequence. Our newly implemented ﬁltering program eﬀectively removed suchvariations and identiﬁed 117 line-speciﬁc mutations,which are likely to be caused by heavy-ion beam irradiations. PCR and sequencing analysis showed thatamong the randomly-chosen 87 loci, 85 had the deﬁnedmutations. These results indicates that the process
for narrowing down the candidates, which was implemented in the pipeline is reasonable and greatly improves the eﬃciency, as conﬁrmed in subsequent experiments. This pipeline is a fundamental resource for
rapid, comprehensive, and cost-eﬀective genome-wide
mutation screening and analysis of rice.

--Fig. 1. A flow chart of the developed bioinformatics
pipeline for exome sequencing datasets

References
1) H. Li and R. Durbin: Bioinformatics 25, 1754–60(2009).
2) A. McKenna et al.: Genome Res. 20, 1297–1303 (2010).
3) K. Ye et al.: Bioinformatics 25, 2865-28-71 (2009).
4) A. R. Quinlan and I. M. Hall: Bioinformatics 26, 841842 (2010).
5) S. Andrews: available from http://www.bioinformatics.
babraham.ac.uk/projects/fastqc.