RIKEN Integrated Sequence Analysis (RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer (original) (raw)

  1. Kazuhiro Shibata1,10,
  2. Masayoshi Itoh1,10,
  3. Katsunori Aizawa1,
  4. Sumiharu Nagaoka1,8,
  5. Nobuya Sasaki1,9,
  6. Piero Carninci1,
  7. Hideaki Konno1,
  8. Junichi Akiyama1,
  9. Katsuo Nishi1,
  10. Tokuji Kitsunai2,
  11. Hideo Tashiro2,
  12. Mari Itoh1,
  13. Noriko Sumi1,
  14. Yoshiyuki Ishii1,
  15. Shin Nakamura1,
  16. Makoto Hazama1,
  17. Tsutomu Nishine1,
  18. Akira Harada1,
  19. Rintaro Yamamoto1,
  20. Hiroyuki Matsumoto1,
  21. Sumito Sakaguchi1,
  22. Takashi Ikegami1,
  23. Katsuya Kashiwagi1,
  24. Syuji Fujiwake1,
  25. Kouji Inoue1,
  26. Yoshiyuki Togawa1,
  27. Masaki Izawa1,4,
  28. Eiji Ohara4,
  29. Masanori Watahiki4,
  30. Yuko Yoneda4,
  31. Tomokazu Ishikawa1,5,
  32. Kaori Ozawa5,
  33. Takumi Tanaka5,
  34. Shuji Matsuura5,
  35. Jun Kawai1,
  36. Yasushi Okazaki1,
  37. Masami Muramatsu1,
  38. Yorinao Inoue6,
  39. Akira Kira7, and
  40. Yoshihide Hayashizaki1,3,11
  41. 1Genome 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), Tsukuba-shi, Ibaraki, 305–0074, Japan; 2Advanced Engineering Center, RIKEN, Wako-shi, Saitama, 351–0198, Japan; 3Medical School, Tsukuba University, Tsukuba-shi, Ibaraki 305–8575, Japan; 4Research & Development, Nippon Gene Co., Ltd., Arakawa, Toyama, 930–0834, Japan; 5Osaka Research Laboratories, Wako Pure Chemical Industries, Ltd., Amagasaki, Hyogo 661–0963, Japan; 6RIKEN Harima Institute at SPring-8, Sayou, Hyougo 679–5148, Japan;7RIKEN, Wako-shi, Saitama, 351–0198, Japan

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.

Footnotes