The document discusses an RNA-seq workshop agenda with the following key points: 1) It includes sessions on RNA-seq introduction, analysis workflows from mapping to differential expression and enrichment analysis, and available online resources and tools for RNA-seq data analysis. 2) Differential expression analysis will be performed using DESeq2 and genes will be analyzed for enrichment in Gene Ontology terms and KEGG pathways to identify biological processes and pathways impacted. 3) The workshop aims to introduce common RNA-seq analysis concepts and provide hands-on experience with tools like DESeq2, GO and KEGG enrichment to find differentially expressed genes and interpret results biologically.

1. S
16:00-17:00
2018 6/20 (Wed.)
T
()
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2. (NGS )
[MA/NGS]
Enrichment (GO, pathway), (heatmap, etc)
(Differential Expressed Genes: DEG) [MA/NGS]

3. (NGS )
[MA/NGS]
Enrichment (GO, pathway), (heatmap, etc)
(Differential Expressed Genes: DEG) [MA/NGS]

4. R
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http://tylervigen.com/spurious-correlations

7. )
(
][
[
https://www.nature.com/articles/ncomms9727

8. ( )
SVM,
( )PCA, t-SNE ( )

9. -- [ ]
-- [ ] R and Bioconductor / command line / python
-- [ ]
-- [ ] ( )
-- [ ] (2 1 )
-- [ ] 29 NGS
https://biosciencedbc.jp/human/human-resources/workshop/h29
-- [ ] RNA-Seq ( )
-- [ ] Dr. Bono
-- [ ] ( )
script
shinzy.nakaoka@gmail.com

10. Online course
Stanford ( )
QIITA ( / )
Stack Overflow ( / )
Udemy
90%
R, python
( )
python/R

11. (NGS )
[MA/NGS]
Enrichment (GO, pathway), (heatmap, etc)
(Differential Expressed Genes: DEG) [MA/NGS]

12. RNA-seq introduction
RNA-seq flow
RNA (cDNA )
(PCR )
( )
Z Wang et al, RNA-Seq: a revolutionary tool for
transcriptomics (2009)

13. RNA-seq hr
RNA-seq hr
Selected genes
by edgeR, DESeq2
(RNA-seq) and
limma,
RankProduct
(microarray)
BAM2ReadCount
Gene α
Gene ω
Gene β
ŸŸŸ
Gene α
Gene ω
Gene β
Time-course visualization
Enrichment
Clustering
Network
inference
i
Keratinization
⏎
GO/KEG
G
PPI, pathway
R Thr
I TS PG ka
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P ncqibgs
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14. D mlu o D d R bE
n moV
Lamontagne J, Mell JC, Bouchard MJ (2016) Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes
to Normal Hepatocyte Gene Expression. PLoS Pathog 12(2): e1005438. doi:10.1371/journal.ppat.1005438
Accession number: GSE68113
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15. Assembly, mapping
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16. Quality Check
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17. Triming
- A d ni -
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Gene001 958
Gene002 7
Gene003 491
Gene004 1649
Gene005 1187
Gene006 12
Gene001 492
Gene002 4
Gene003 246
Gene004 861
Gene005 620
Gene006 7
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18. (Spliced) mapping
Tophat / Bowtie2 / subread hps (spliced) mapping)
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19GGAE jP L E )CA ED E 19G 9D n j h ts
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GCA BLE A E u o TpUhgcdTs
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19. Alignment
STAR S ultra-fast alignment
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20. : DESeq2 ( ) ( ) MA-plot (M: A )
https://www.bioconductor.org/packages/devel/bioc/vignett
es/DESeq2/inst/doc/DESeq2.pdf

21. Affymetrix Gene Chip 1 Gene Expression Omnibus
(Agilent ) (Cyanin dyes Cy3: Cy5: )
background noise (
)
: Mas5 (Affy), RMA (Affy), GCRMA (Affy), LOWESS (Agilent), QuanJle (all)
NGS
short read maping
transcript
GC ( PCR )
: TMM (edgeR), DEGSeq2, RPKM, FPKM (Cufflinks), Median, Quantile etc…

22. (
(replicate )
23000
FDR (False Discovery Ratio)
(Benjamini and Hochberg)
: limma (linear model of microarray), rank product
NGS
Read count (short read reference genome
) RPKM Read count (
Poisson )
: edgeR,, DEGSeq2, cuffdiff, voom (RPM limma ) etc…
( )

23. (Uninfected vs Ad-Infected)
: DEGSeq2 ( )
Fabp1 9.141085127 14.16261662 7.16E-32 5.25E-36
G6pc 9.308228996 13.12791835 3.23E-29 4.73E-33
Gnmt 7.972094667 10.85576105 1.19E-28 2.62E-32
Stac3 8.512799293 11.07394801 2.72E-28 7.99E-32
Sult1c3 8.598851154 10.83051428 4.04E-28 1.48E-31
Cyp3a2 8.643053186 13.34346161 1.19E-27 5.22E-31
Cyp3a18 7.73011204 12.40583051 3.34E-26 1.71E-29
Cyp2c7 8.080204091 14.13579037 4.41E-26 2.59E-29
Slc10a1 7.251633979 11.9006561 1.23E-25 8.14E-29
Ftcd 7.242311435 11.19173058 1.25E-24 9.15E-28
Hsd3b5 7.769501627 9.64088714 1.80E-24 1.45E-27
Cyp8b1 8.022740425 10.21022277 3.20E-24 2.82E-27
Cyp2e1 7.990226129 13.24331587 1.10E-23 1.05E-26
Ttc36 7.857192495 9.257031182 1.17E-23 1.21E-26
RGD1307603 7.384176218 11.04351261 1.50E-23 1.65E-26
Serpind1 6.718175421 12.53716856 9.42E-22 1.10E-24
Bhmt 6.386565955 14.36571852 1.08E-21 1.35E-24
Ust5r 6.387315677 10.25543675 2.74E-21 3.62E-24
Thrsp 6.080443377 11.76462419 3.68E-21 5.13E-24
Cyp3a2 - Cytochrome
P450 3A2

24. Enrichment
Enrichment ( )
STAT3 ( ) GO terms
Gene Ontology MF (Molecular Function), CC (Cellular Component),
BP (Biological Process) (ontology)
GO ID Qualified GO term Evidence PubMed IDs
GO:0000981 RNA polymerase II
transcription factor activity,
sequence-specific DNA
binding
IEA
GO:0004871 signal transducer activity IEA,TAS 7512451
GO:0005515 protein binding IPI 8662591
GO:0008134 transcription factor binding IPI 15664994
GO:0046983 protein dimerization activity ISS
GO ID Qualified GO term Evidence PubMed IDs
GO:0005739 mitochondrion IEA
GO:0090575 RNA polymerase II
transcription factor complex
IMP 16946298

25. Enrichment
Enrichment ( )
(DEG)
STAT3, JAK2, NFkB, IRF4, CRCX12, CCR5, IL1b etc…
GO:0019221 (cytokine-mediated signaling pathway) GO term
GO:0019221
GO:0019221
N
n K k
(GO term)
DEG (GO term)
p
Gene Ontology DEG (
) Gene Set Enrichment Analysis
(parametric GSEA)

26. Enrichment
(GO enrichment)
DEGSeq2 Gene Ontology (MF/BP/CC)
KEGG pathway, Reactome pathway ontology pathway
Gene Ontology (Biological Process)
GO:0006082 organic acid metabolic process 1.76E-22
Ehhadh/Adh7/Adh1/Cyp2d3/Cyp4a2/Ddc/Fabp1/Fbp1/Agxt/Cyp2a2/Sds/Cyp2e1/
Rgn/Gnmt/Acox2/Otc/G6pc/Ugt2b1/Cyp2c7/Glyat/Cyp2c6v1/Baat/Cyp4a3/Hgd/P
ck1/Bhmt2/Cyp4a1/Kmo/Slc27a2/Marc1/Slc27a5/Bhmt/Abat/Cdo1/Hao2/Ftcd
GO:0043436 oxoacid metabolic process 6.61E-22
Ehhadh/Adh7/Adh1/Cyp2d3/Cyp4a2/Ddc/Fabp1/Fbp1/Agxt/Cyp2a2/Sds/Cyp2e1/
Rgn/Gnmt/Acox2/Otc/Ugt2b1/Cyp2c7/Glyat/Cyp2c6v1/Baat/Cyp4a3/Hgd/Pck1/B
hmt2/Cyp4a1/Kmo/Slc27a2/Marc1/Slc27a5/Bhmt/Abat/Cdo1/Hao2/Ftcd
GO:0019752 carboxylic acid metabolic process 8.77E-22
Ehhadh/Adh7/Adh1/Cyp2d3/Cyp4a2/Ddc/Fabp1/Fbp1/Agxt/Cyp2a2/Sds/Cyp2e1/
Rgn/Gnmt/Acox2/Otc/Ugt2b1/Cyp2c7/Glyat/Cyp2c6v1/Baat/Cyp4a3/Hgd/Pck1/B
hmt2/Cyp4a1/Kmo/Slc27a2/Slc27a5/Bhmt/Abat/Cdo1/Hao2/Ftcd
GO:0044281 small molecule metabolic process 3.24E-20
Ehhadh/Adh7/Adh1/Cat/Cyp2d3/Cyp4a2/Ddc/Fabp1/Fbp1/Hsd3b5/Lcat/Agxt/Cyp
2a2/Sds/Cyp2e1/Rgn/Gnmt/Acox2/Otc/G6pc/Rbp4/Cyp3a2/Ugt2b1/Cyp2c7/Glyat
/Cyp2c6v1/Baat/Cyp4a3/Hgd/Pck1/Bhmt2/Apof/Cyp4a1/Car5a/Kmo/Slc27a2/Mar
c1/Slc27a5/Bhmt/Abat/Cdo1/Hao2/Ftcd
GO:0032787 monocarboxylic acid metabolic process 4.05E-19
Ehhadh/Adh7/Adh1/Cyp2d3/Cyp4a2/Fabp1/Fbp1/Agxt/Cyp2a2/Sds/Cyp2e1/Rgn/
Acox2/Ugt2b1/Cyp2c7/Glyat/Cyp2c6v1/Baat/Cyp4a3/Pck1/Cyp4a1/Kmo/Slc27a2/
Slc27a5/Abat/Hao2/Ftcd
GO:0044710 single-organism metabolic process 8.60E-18
Ehhadh/Adh7/Adh1/C4bpb/C6/Cat/Cyp2d3/Cyp4a2/Ddc/Fabp1/Fbp1/Hsd3b5/Lca
t/Mbl1/Agxt/Cyp2a2/Sult2a1/Sds/Cyp2e1/Rgn/Gnmt/Acox2/Cyp3a18/Thrsp/Crp/
Otc/G6pc/Rbp4/Cyp3a2/Ugt2b1/Ces1e/Cyp2c7/Glyat/Cyp2c6v1/Baat/Cyp4a3/Dhr
s7/Serpina6/Hgd/Pck1/Adhfe1/Bhmt2/Aox3/RGD1564865/Apof/Cyp4a1/Slco1a1/
Car5a/Kmo/Slc27a2/Marc1/Slc27a5/Bhmt/Abat/Cdo1/Cyp8b1/Hao2/Ftcd
GO:0055114 oxidation-reduction process 1.19E-16
Ehhadh/Adh7/Adh1/Cat/Cyp2d3/Cyp4a2/Fabp1/Hsd3b5/Cyp2a2/Cyp2e1/Gnmt/A
cox2/Cyp3a18/G6pc/Cyp3a2/Cyp2c7/Cyp2c6v1/Cyp4a3/Dhrs7/Hgd/Adhfe1/Aox3/
RGD1564865/Cyp4a1/Kmo/Slc27a2/Marc1/Cdo1/Cyp8b1/Hao2
GO:0006629 lipid metabolic process 2.78E-14
Ehhadh/Adh7/Adh1/Cat/Cyp2d3/Cyp4a2/Fabp1/Hsd3b5/Lcat/Cyp2a2/Sult2a1/Cy
p2e1/Rgn/Acox2/Thrsp/G6pc/Rbp4/Cyp3a2/Ces1e/Cyp2c7/Cyp2c6v1/Baat/Cyp4a
3/Serpina6/Pck1/Apof/Cyp4a1/Slc27a2/Slc27a5/Hao2
GO:0044255 cellular lipid metabolic process 1.54E-13
Ehhadh/Adh7/Adh1/Cat/Cyp2d3/Cyp4a2/Fabp1/Lcat/Cyp2a2/Cyp2e1/Rgn/Acox2/
Thrsp/G6pc/Rbp4/Cyp3a2/Cyp2c7/Cyp2c6v1/Baat/Cyp4a3/Pck1/Apof/Cyp4a1/Slc
27a2/Slc27a5/Hao2

27. Enrichment
(Reactome enrichment)
DEGSeq2 Gene Ontology (MF/BP/CC)
KEGG pathway, Reactome pathway ontology pathway

28. −1
0
1
2
0 5 10 15 20
AM
Gene
nonDEG
DEG
Dlx3
(DEG) ( )
MA-plot GO bar plot
MA-plot
hair cell differentiation
inner ear receptor cell development
detection of mechanical stimulus involved in sensory perception
multicellular organismal process
single−multicellular organism process
detection of mechanical stimulus involved in sensory perception of sound
fibril organization
neuromuscular process
extracellular fibril organization
neuromuscular process controlling balance
0.0 0.5 1.0 1.5 2.0
Q value (−log10)
DEG
Enriched
GO-barplot

29. (heatmap)
qplot z
heatmap.2 (>500)
(eps) heatmap.2 option
( ) ggplot2 heatmap.2

30. Tips
Q. ( )
Q. (UCSC/Emsemble)
Q.
Q.
Q.
Q. Read replicate
Q. mRNA ( ) polyA

31. RNA-seq
Brief Bioinform (2013) 14 (6): 671-683.doi:
10.1093/bib/bbs046
edgeR
TMM (Trimmed Mean of M-
value) DESeq
NCBI SRA
(FASTQ sra )
(RPKM)
Count data edgeR or
DESeq2 or voom (limma)
, cuffdiff RPKM

32. 1
RNA-seq annotation ( (
Zhao S, Zhang B. A comprehensive evaluation of ensembl, RefSeq, and UCSC
annotations in the context of RNA-seq read mapping and gene quantification. BMC
Genomics. 2015, 16:97
NCBI / Ensembl / UCSC (
1 Human Body
Map 2.0 Project RNA-seq
Ensemble ) 50% ( 6 unique ( )
( RefSeq vs Ensemble 6

33. RNA. 2016 Jun; 22(6): 839–851. doi: 10.1261/rna.053959.115
n=3 (biological replicates)
(edgeR / DESeq2
/ Cuffdiff etc)
fold-change threshold
Fold-change
n=6

34. Independent filtering
www.pnas.org/cgi/doi/10.1073/pnas.0914005107
http://master.bioconductor.org/help/course-
materials/2009/BioC2009/talks/Independent_filters_and_multiple_testing.pdf
( ) θ % t
Type I error filtering

35. ComBat
W.E. Johnson, C. Li, and A. Rabinovic. Adjusting batch effects in microarray data
using empirical bayes methods. Biostatistics, 8(1):118–127, 2007.
: Caleb K Stein et al, Blood 2014 124:3355; Fig.1
replicate
ComBat sva package
ComBat

36. replicate
RNA-seq differential expression studies: more sequence or more replication?
Vol. 30 no. 3 2014, pages 301–304 doi:10.1093/bioinformatics/btt688
10M
replicate
(
depth
replicate
)

37. mRNA
mRNA polyA
Scientific Reports | (2018) 8:4781 | DOI:10.1038/s41598-018-23226-4
Ribosomal RNA (rRNA) robozero polyA mRNA
(human blood, colon) RNA Ribozero
( ) protein-coding RNA ( )
PolyA protein-coding RNA

38. Memo

39. 40
(DNA chip) , ( )
DNS (cDNA)
short read
mRNA tRNA, 99%
PCR
Affymetrix, Agilent, Illumina ( )
PCR, Roche FLX, Ion PGM bridge PCR, Illumina Hiseq (single/pair end),
: PacBio (long-read), Oxford Nanopore
(depth),
Quality check (RIN ) GC ,
CEL
DEG (Differentially Expressed Genes) GO (Gene Ontology)
mapping ( ), bowtie2&tophat2
FASTA&FASTQ, , BAM, count-data, RPKM ( )
de novo ( )
Gene set enrichment
, heatmap, , (False Discovery Ratio)
log-normal
PCR bias, single cell
R & Bioconductor NCBI/EMBL /DDBJ BLAST, GEO, SRA

40. https://www.genome.gov/13014330/transcriptome-fact-sheet/
Gene+ome ( ) = genome
Transcript ( )
= transcriptome
(microarray NGS)
( )
(
)

41. https://www.genome.gov/10000533/dna-microarray-technology/
DNA
Chip
( )
DNA
RNA
mRNA
cDNA
( )
( )

42. PCR
https://www.genome.gov/10000207/pcr-fact-sheet/
PCR (Polymerase Chain Reaction)
( ) DNA
DNA (DNA )
DNA
( )
DNA
DNA
DNA
DNA

43. http://bitesizebio.com/13546/sequencing-by-synthesis-
explaining-the-illumina-sequencing-technology/
cDNA
(50-100bp ) PCR
DNA
(ligate)
DNA
( )
(bridge amplification)
(Roche Illumina)
Illumina (bridge PCR)
Maxiam-Gilbert Sanger Nanopore
DNA
(4 )
(sequencing by synthesis)

44. http://bitesizebio.com/13546/sequencing-by-synthesis-
explaining-the-illumina-sequencing-technology/
DNA
de novo ( )
SNP (variant
call) : Trinity, Velvet, BWA
de novo
Exome
exon ( )
(Picard) SNV (Single Nucleotide
Variation)/Indel (snpEff)
DNA

45. https://en.wikipedia.org/wiki/Methylated_DNA_immunop
recipitation
(Chromatin ImmunoPrecipitation) DNA
RNA
CLIP (Cross-Linking ImmunoPrecipitation)-seq
ChIP-seq
Methylated DNA immunoprecipitation (MeDIP) seq
5-methylcytosine (5mC) DNA
(Immunoprecipitation)
(distal)
Chromosome
Confirmation Capture (3C) Hi-C
ENCODE project

46. http://www.nias.affrc.go.jp/gmogmo/FAQ/app/J3.html
16S rRNA
sequencing
16S rRNA ( )
Whole Genome Shotgun (WGS) sequence
alignment
WGS
de novo
WGS (fragment)
(assembly) contig (contiguous
sequence) contig scaffold
( )

47. http://www.nias.affrc.go.jp/gmogmo/FAQ/app/J3.html
( )
Mouse Phenome Database (MPD)
http://phenome.jax.org/