An advantage of genomics is the development of a next-generation sequencer that can decode DNA sequences at the giga level. Development of the next-generation DNA sequencer was advanced under the Affordable Care Act aims to realize societal implementation of medical genomics [27, 28]. The desktop-type next-generation sequencer MiSeq has the ability to read 15 million DNA sequences in one run. Generally, whole-genome sequencing analysis required 30 times of the given genome size (30× genome coverage). According to this standard scale, the MiSeq treats only one rice sample per run, because the 15 million DNA amount is just enough to reconstruct a single rice genome. However, it is a big problem that the running cost is excessively high to use the MiSeq at the practical breeding to detect target genes. So, in this study, to achieve a minimum scale to detect target genes with a reasonable cost, we try to detect Jukkoku_sd1 at only 5× rice genome coverage by using the MiSeq. The semidwarf gene sd1 (a loss-of-function mutation of the GA20-oxidase encoding gene) was transferred to Koshihikari by consecutive backcrosses to prepare a semidwarf Koshihikari named Koshihikari Jukkoku_sd1. The Koshihikari Jukkoku_sd1 backcross was used to detect single-nucleotide polymorphisms (SNPs) in Jukkoku-derived sd1 by NGS. Whole-genome analysis was conducted using Koshihikari Jukkoku_sd1 and Koshihikari. Genomic DNA was extracted from each cultivar by the CTAB (hexadecyltrimethylammonium bromide) method. Genomic DNA was tagged and fragmented to an average length of 500 bp using the Nextera® transposome-based approach. After purification of the transposome with DNA Clean & Concentrator™-5 (Zymo Research, Irvine, CA, USA), adaptors for fixation on the flow cell were synthesized at both ends of each fragment using a polymerase chain reaction (PCR). Then, the DNA fragments were subjected to size selection using AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA). Finally, qualitative and quantitative measurements were performed using a Fragment Analyzer™ (Advanced Analytical Technologies, Inc., Ankeny, IA, USA) and a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) to prepare a DNA library for NGS. Aiming to achieve 5× rice genome coverage, a MiSeq next-generation sequencer was used to simultaneously analyze five lines; namely, 4-5 ng of five DNA libraries was applied in each MiSeq run. Clusters then were formed on the flow cells by bridge-PCR and each pair-end of a 250 bp read was sequenced. Resulting sequenced reads were mapped using BWA software with the Nipponbare genome  as a reference. Then, SNPs and Indels were detected using SAMtools software (http://samtools.sourceforge.net/).