title: RIKEN integrated sequence analysis (RISA) system--384-format sequencing pipeline with 384 multicapillary sequencer.
abstract: The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3' end and 5' end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month.
authors.affiliation: Genome Exploration Research Group, RIKEN, Genomic Sciences Center (GSC) and Genome Science Laboratory, RIKEN, Tsukuba Institute, Core Research of Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Ibaraki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Shibata
authors.forename: M
authors.initials: M
authors.lastname: Itoh
authors.forename: K
authors.initials: K
authors.lastname: Aizawa
authors.forename: S
authors.initials: S
authors.lastname: Nagaoka
authors.forename: N
authors.initials: N
authors.lastname: Sasaki
authors.forename: P
authors.initials: P
authors.lastname: Carninci
authors.forename: H
authors.initials: H
authors.lastname: Konno
authors.forename: J
authors.initials: J
authors.lastname: Akiyama
authors.forename: K
authors.initials: K
authors.lastname: Nishi
authors.forename: T
authors.initials: T
authors.lastname: Kitsunai
authors.forename: H
authors.initials: H
authors.lastname: Tashiro
authors.forename: M
authors.initials: M
authors.lastname: Itoh
authors.forename: N
authors.initials: N
authors.lastname: Sumi
authors.forename: Y
authors.initials: Y
authors.lastname: Ishii
authors.forename: S
authors.initials: S
authors.lastname: Nakamura
authors.forename: M
authors.initials: M
authors.lastname: Hazama
authors.forename: T
authors.initials: T
authors.lastname: Nishine
authors.forename: A
authors.initials: A
authors.lastname: Harada
authors.forename: R
authors.initials: R
authors.lastname: Yamamoto
authors.forename: H
authors.initials: H
authors.lastname: Matsumoto
authors.forename: S
authors.initials: S
authors.lastname: Sakaguchi
authors.forename: T
authors.initials: T
authors.lastname: Ikegami
authors.forename: K
authors.initials: K
authors.lastname: Kashiwagi
authors.forename: S
authors.initials: S
authors.lastname: Fujiwake
authors.forename: K
authors.initials: K
authors.lastname: Inoue
authors.forename: Y
authors.initials: Y
authors.lastname: Togawa
mesh_terms: D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000191:economics / Q000295:instrumentation* / Q000379:methods* / Q000592:standards; D004591:Electrophoresis, Polyacrylamide Gel / Q000191:economics / Q000295:instrumentation / Q000379:methods / Q000592:standards; D016678:Genome; D019044:Image Cytometry / Q000191:economics / Q000295:instrumentation / Q000379:methods / Q000592:standards; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics / Q000295:instrumentation* / Q000379:methods* / Q000592:standards; D013698:Templates, Genetic
chemical_list: D004247:DNA