The rice reference genome revisited: extensive updateby the PacBio SMRT sequencing technology
Takeshi Itoh, Yoshihiro Kawahara, Tomoko Hirozane-Kishikawa, Hiroaki Sakai

Bioinformatics Team, Advanced Analysis Center, National Agriculture and Food Research Organization
2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan
Since its publication in 2005, the rice reference genome of cv. Nipponbare has widely been an indispensable research foundation ofgenome-wide studies for rice and other cereal crops. An advantage to use this reference genome lies in the fact that it was basedon a physical map of BAC/PAC clones and determined by the Sanger method, so that the resultant sequences are highly accurate.
Later, the advent of the era of high-throughput sequencing required an even more accurate genome sequence to be compered.
Therefore, the rice reference genome was thoroughly reexamined by using a large number of Illumina reads and a novel highquality genome assembly, IRGSP-1.0 was released in 2013. However, since the genome still remained incomplete because ofhundreds of gaps, we decided to fill them by PacBio's single molecule real-time sequencing technology that can produce long-readsequences. To close the gaps of IRGSP-1.0, we used ~13x coverage PacBio reads and PBJelly. As the PacBio reads are highly
error-prone, the gaps filled by PacBio reads as well as the other reference genome regions were validated by ~40x Illumina reads.
As a result, the number of the gaps was reduced from 558 to 255 and the total genome length increased by more than 1 Mb. In
addition, thousands of errors were corrected by the Illumina reads. It is anticipated that the release of this new rice reference
genome will expedite omics researches of rice and closely related species.

The genome of a japonica rice cultivar, Nipponbare, wasdeciphered in 2005. It is based on a BAC/PAC physicalmap and highly accurate, but there remained severalgaps.

Sequenced Telomer

In 2013 the genome sequence wasimproved by using Illumina and 454
reads. However they were not usefulto close the gaps.

We decided to employ PacBio for gap-closing.
PacBio sub-reads
494,889 reads,
5,109,401,763 bp (~13x)
Avg:10,324 bp, Max: 47.3 kb

Gap-closing
by PBJelly2

IRGSP-1.0 (before corrections)

Total Nt(bp)374,424,240# ofScaffolds
61

Ilumina PE100 reads
93,572,213 pairs (~40x)

Error correction
by BWA-MEM& GATK

Two steps to improve the Nipponbare genome (IRGSP-1.0)# ofGaps
558

Total Gap
lengths (bp)101,607

Telomeric Ns(bp)19,118Treated by PBJelly (conducted twice: ≥25 bp gaps and ≥1 bp gaps)

Total Nt(bp)375,432,199# ofScaffolds
57# ofGaps
225

Total Gap
lengths (bp)47,486

Telomeric Ns(bp)
19,101

The total nucleotide length increased by >1 Mb.
The number of gaps decreased by 333 (59.7%).
Error corrections by Illumina reads
Chromosomes
Unanchored contigs

Sequenced Centromeres

The Rice Annotation
Project Database
http://rapdb.dna.affrc.go.jp/

Total Nt
374,160,882
1,271,317

SNV
3,944
383

Insertion
3,765
593

Deletion
3,942
851

After the Illumina readcorrections, a numberof reasonable primarystructures of CDSs
were restored. In thisexample,
twoinsertions led to thecomplete CDS.
• We will further improve the Nipponbare reference genome by using other platforms,
such as Nanopore.
• The data will be released from the RAP-DB (http://rapdb.dna.affrc.go.jp/).
Acknowledgement This work was supported by a grant from the Ministry of Agriculture,
Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement,
IVG2001).