20110602labseminar pub

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TrinityとNewbler2.5を用いて454データに対するde novo transcriptome assembleをしたときの精度比較
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20110602labseminar pub

  1. 1. RNA-Seq (yag_ays)http://yag-ays.jp/pdf/20110602labseminar_pub.pdf
  2. 2. r e d n s o c eusagiusamimi
  3. 3. NGS (Next Generation Sequencing) RNA-Seq (Transcriptome Analysis) de novoTranscriptome Assembly
  4. 4. Central Dogma A T G C DNA mRNA Protein
  5. 5. Central Dogma A T G C DNA mRNA Protein
  6. 6. Central Dogma A T G C DNA Transcriptome mRNA Protein
  7. 7. NGS RNA-SeqA T G C NGS • illumina / Solexa GA • ABI / SOLiD • Roche / 454 • PacBio • Helicos / Heliscope • ion torrent etc...mRNA TTAGCCTTAGCTTCC GTCGCAACTTCCTTA TTCACGAGCTTGATG TTGCGGATCACTTTG
  8. 8. NGS RNA-SeqA T G C NGS NGS • illumina / Solexa GA • • ABI / SOLiD • Roche / 454 • • PacBio • Helicos / Heliscope • ion torrent etc... •mRNA TTAGCCTTAGCTTCC GTCGCAACTTCCTTA TTCACGAGCTTGATG TTGCGGATCACTTTG
  9. 9. RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
  10. 10. RNA-Seq • • • ʻalign-then-assembleʼ ʻassemble-then-aliignʼ approach approach
  11. 11. RNA-Seq • 454 • • ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
  12. 12. RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
  13. 13. RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
  14. 14. RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
  15. 15. RNA-Seq • • cDNA ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
  16. 16. Sujai Kumar and Mark L Blaxter : Comparing de novoassemblers for 454 transcriptome data (2010)Newbler 2.5
  17. 17. Sujai Kumar and Mark L Blaxter : Comparing de novoassemblers for 454 transcriptome data (2010)Newbler 2.5 ...
  18. 18. Sujai Kumar and Mark L Blaxter : Comparing de novoassemblers for 454 transcriptome data (2010)Newbler 2.5 ... Trinity...!!
  19. 19. 1. Newbler 2.5 • Roche 454 • 454 • 2. Trinity • Broad Institute • 454 ( ) • Nat Biotechnol. 2011 May ** Grabherr MG, Haas BJ,Yassour M et al. : Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011 May 15
  20. 20. 1. Newbler 2.5 • Overlap-Layout-Consensus (OLC)2. Trinity I. Inchworm : k-mer graph II. Chrysalis : Contig pool III.Butterfly : De Bruijn Graph 2
  21. 21. Roche 454 pyrosequencing usamimi 0.3M reads (sff or fastq format)Newbler 2.5 Trinity (fasta format) (fasta format) GMAP with usagi CDS (gff format) (gff format)
  22. 22. S. Kumar et al.(2010) • ( ) •
  23. 23. Newbler 2.5 Trinity Newbler 2.5 TrinityNumber of 19,753 20,758 contigsTotal Bases 9,651,390 10,275,166Max contig 2,878 2,151 lengthMean contig 488.6 495 length N50 581 616
  24. 24. Newbler 2.5 N = 19,753 Trinity N = 20,758http://edwards.sdsu.edu/prinseq_beta/
  25. 25. usagi CDS all usagi CDS 30,000 ≧ 80% alignment ≧ 90% alignment ≧ 95% alignment Newbler 2.5 Trinity 100% alignment 16000 all 15,498 15,524 12000≧ 80% 14,583 14,697 8000≧ 90% 8,466 8,665≧ 95% 1,059 1,191 4000100% 66 30 0 Newbler 2.5 Trinity
  26. 26. usagi Newbler 2.5 Trinity12,417 10,433 genes genes 2,990 9,427 1,006
  27. 27. ... S. Kumar et al.(2010) Poly(A/T)Poly(A/T) Poly(A/T)
  28. 28. Poly(A/T) Trinity > Newbler 2.5 Newbler 2.5 Trinity 257 3,773Poly T (1.30%) (18.18%) 20 bp 20 bp 539 2,349Poly A (2.73%) (11.32%) 20 bp 20 bphttp://edwards.sdsu.edu/prinseq_beta/ ()
  29. 29. Poly(A/T) Trinity > Newbler 2.5 Newbler 2.5 Trinity 257 3,773Poly T (1.30%) (18.18%)Poly(A/T) Quality Value 20 bp 20 bp →Newbler Quality trimming ...? 539 2,349Poly A (2.73%) (11.32%) 20 bp 20 bp http://edwards.sdsu.edu/prinseq_beta/ ()
  30. 30. Trinity Newbler 2.51 usagi CDS2 Poly(A/T) Trinity Newbler 2.5
  31. 31. Trinity Newbler 2.51 usagi CDS Trinity 4542 Poly(A/T) Trinity Newbler 2.5
  32. 32. Trinity Newbler 2.51 usagi CDS Trinity 4542 Poly(A/T) Trinity Newbler 2.5
  33. 33. Method : Parameters• Newbler 2.5 • Trinity (20110519 ver.) • -notrim • --seqType=fq • -urt • --single • --min_contig_length 50 • --run_butterfly • --CPU 4 • --bfly_opts "-- compatible_path_extensi on --stderr "

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