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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

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
@瀬々ラボセミナー

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    20110602labseminar pub 20110602labseminar pub Presentation Transcript

    • RNA-Seq (yag_ays)http://yag-ays.jp/pdf/20110602labseminar_pub.pdf
    • r e d n s o c eusagiusamimi
    • NGS (Next Generation Sequencing) RNA-Seq (Transcriptome Analysis) de novoTranscriptome Assembly
    • Central Dogma A T G C DNA mRNA Protein
    • Central Dogma A T G C DNA mRNA Protein
    • Central Dogma A T G C DNA Transcriptome mRNA Protein
    • 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
    • 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
    • RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
    • RNA-Seq • • • ʻalign-then-assembleʼ ʻassemble-then-aliignʼ approach approach
    • RNA-Seq • 454 • • ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
    • RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
    • RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
    • RNA-Seq ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
    • RNA-Seq • • cDNA ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
    • Sujai Kumar and Mark L Blaxter : Comparing de novoassemblers for 454 transcriptome data (2010)Newbler 2.5
    • Sujai Kumar and Mark L Blaxter : Comparing de novoassemblers for 454 transcriptome data (2010)Newbler 2.5 ...
    • Sujai Kumar and Mark L Blaxter : Comparing de novoassemblers for 454 transcriptome data (2010)Newbler 2.5 ... Trinity...!!
    • 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
    • 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
    • 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)
    • S. Kumar et al.(2010) • ( ) •
    • 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
    • Newbler 2.5 N = 19,753 Trinity N = 20,758http://edwards.sdsu.edu/prinseq_beta/
    • 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
    • usagi Newbler 2.5 Trinity12,417 10,433 genes genes 2,990 9,427 1,006
    • ... S. Kumar et al.(2010) Poly(A/T)Poly(A/T) Poly(A/T)
    • 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/ ()
    • 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/ ()
    • Trinity Newbler 2.51 usagi CDS2 Poly(A/T) Trinity Newbler 2.5
    • Trinity Newbler 2.51 usagi CDS Trinity 4542 Poly(A/T) Trinity Newbler 2.5
    • Trinity Newbler 2.51 usagi CDS Trinity 4542 Poly(A/T) Trinity Newbler 2.5
    • 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 "