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裕樹 奥田
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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 @瀬々ラボセミナー
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20110602labseminar pub
1.
RNA-Seq
(yag_ays) http://yag-ays.jp/pdf/20110602labseminar_pub.pdf
2.
r e d
n s o c e usagi usamimi
3.
NGS (Next
Generation Sequencing) RNA-Seq (Transcriptome Analysis) de novo Transcriptome Assembly
4.
Central Dogma
A T G C DNA mRNA Protein
5.
Central Dogma
A T G C DNA mRNA Protein
6.
Central Dogma
A T G C DNA Transcriptome mRNA Protein
7.
NGS
RNA-Seq A T G C NGS • illumina / Solexa GA • ABI / SOLiD • Roche / 454 • PacBio • Helicos / Heliscope • ion torrent etc... mRNA TTAGCCTTAGCTTCC GTCGCAACTTCCTTA TTCACGAGCTTGATG TTGCGGATCACTTTG
8.
NGS
RNA-Seq A T G C NGS NGS • illumina / Solexa GA • • ABI / SOLiD • Roche / 454 • • PacBio • Helicos / Heliscope • ion torrent etc... • mRNA TTAGCCTTAGCTTCC GTCGCAACTTCCTTA TTCACGAGCTTGATG TTGCGGATCACTTTG
9.
RNA-Seq ʻalign-then-assembleʼ
ʻassemble-then-alignʼ approach approach
10.
RNA-Seq
• • • ʻalign-then-assembleʼ ʻassemble-then-aliignʼ approach approach
11.
RNA-Seq • 454
• • ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
12.
RNA-Seq ʻalign-then-assembleʼ
ʻassemble-then-alignʼ approach approach
13.
RNA-Seq ʻalign-then-assembleʼ
ʻassemble-then-alignʼ approach approach
14.
RNA-Seq ʻalign-then-assembleʼ
ʻassemble-then-alignʼ approach approach
15.
RNA-Seq
• • cDNA ʻalign-then-assembleʼ ʻassemble-then-alignʼ approach approach
16.
Sujai Kumar and
Mark L Blaxter : Comparing de novo assemblers for 454 transcriptome data (2010) Newbler 2.5
17.
Sujai Kumar and
Mark L Blaxter : Comparing de novo assemblers for 454 transcriptome data (2010) Newbler 2.5 ...
18.
Sujai Kumar and
Mark L Blaxter : Comparing de novo assemblers for 454 transcriptome data (2010) Newbler 2.5 ... Trinity...!!
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.
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.
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.
S. Kumar et
al.(2010) • ( ) •
23.
Newbler 2.5 Trinity
Newbler 2.5 Trinity Number of 19,753 20,758 contigs Total Bases 9,651,390 10,275,166 Max contig 2,878 2,151 length Mean contig 488.6 495 length N50 581 616
24.
Newbler 2.5
N = 19,753 Trinity N = 20,758 http://edwards.sdsu.edu/prinseq_beta/
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 4000 100% 66 30 0 Newbler 2.5 Trinity
26.
usagi
Newbler 2.5 Trinity 12,417 10,433 genes genes 2,990 9,427 1,006
27.
...
S. Kumar et al.(2010) Poly(A/T) Poly(A/T) Poly(A/T)
28.
Poly(A/T)
Trinity > Newbler 2.5 Newbler 2.5 Trinity 257 3,773 Poly T (1.30%) (18.18%) 20 bp 20 bp 539 2,349 Poly A (2.73%) (11.32%) 20 bp 20 bp http://edwards.sdsu.edu/prinseq_beta/ ()
29.
Poly(A/T)
Trinity > Newbler 2.5 Newbler 2.5 Trinity 257 3,773 Poly T (1.30%) (18.18%) Poly(A/T) Quality Value 20 bp 20 bp →Newbler Quality trimming ...? 539 2,349 Poly A (2.73%) (11.32%) 20 bp 20 bp http://edwards.sdsu.edu/prinseq_beta/ ()
30.
Trinity Newbler 2.5 1
usagi CDS 2 Poly(A/T) Trinity Newbler 2.5
31.
Trinity Newbler 2.5 1
usagi CDS Trinity 454 2 Poly(A/T) Trinity Newbler 2.5
32.
Trinity Newbler 2.5 1
usagi CDS Trinity 454 2 Poly(A/T) Trinity Newbler 2.5
33.
34.
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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