RIKEN integrated sequence analysis (RISA) system-384-format sequencing pipeline with 384 multicapillary sequencer

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) incl uding a 384-multicapillary sequencer (the so-called RISA sequencer) was dev eloped For the RIKEN mouse encyclopedia project. The RISA system consists o f colony picking, template preparation, sequencing reaction, and the sequen cing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This sys tem consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384 -multicapillary array assembler (CAS), and a 384-multicapillary casting dev ice. The RISA sequencer can simultaneously analyze 384 independent sequenci ng products. The optical system is a scanning system chosen after careful c omparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluoresc ent-labeled sequencing reaction (chain termination reaction), including tra nscriptional sequencing based on RNA polymerase, which was originally devel oped by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully an alyzed 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 seque ncing 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 comb ination of these technologies allowed us to construct the RISA system consi sting 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, mo use, rat domestic animals, and plants, can be revealed by seven RISA system s within one month.