rna-seq at jgiusermeeting.jgi.doe.gov/wp-content/uploads/sites/2/2016/...2016/04/06 · rna-seq...
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RNA-Seq at JGI
Overview of RNA-Seq products
• Transcriptomeassembly
• Differen3algeneexpression
• smallRNA
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GeneExpressionStudies
Howmany?
EmpiricalsupportinGenomeAnnota3onHowdotheysplice?
GeneRegula3onStudies
mRNAcleavage?
AAAA
RNA-Seq Science Programs
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IX
RNA-Seq Workflow
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RNA Samples
RNA Library Construction
Data QC
Genome Annotation
Differential Expression
Data to User
Small RNA Analysis
RNA-Seq Workflow
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RNA Samples
RNA Library Construction
Data QC
Genome Annotation
Differential Expression
Data to User
Small RNA Analysis
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Contamination Check: Phylogeny of Reads vs nt
RNA-Seq Data QC
Average Quality by Base position
Avg
Qua
lity
Scor
e
Read Position
Read Quality > Q30 at Cycle 140
QC Report
RNA-Seq Library QC – Usable reads
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0
10
20
30
40
50
60
70
80
90
100
TTOU TTPX OYHS OYHG OAUW OYHH OYHB ONSB OYHY ONSA
Trans Mapped
rRNA
Artifact
Example Fungal Libraries
Perc
ent R
eads
Non-usable <5%
Mapped >80%
RNA-Seq Workflow
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RNA Samples
RNA Library Construction
Data QC
Genome Annotation
Differential Expression
Data to User
Small RNA Analysis
de novo Assembly - Trinity
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Complex RNA Sample
Reads
de novo Assembly (Trinity)
Assembled Transcriptome Aligned to genome
Read pre- Processing, Normalization
RNA-Seq Workflow
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RNA Samples
RNA Library Construction
Data QC
Genome Annotation
Differential Expression
Data to User
Small RNA Analysis
RNA – Differential Gene Expression
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ConditionA ConditionB BIOLOGICAL REPLICATES!
Align reads to genome = HISAT
Read count = featureCounts
Normalize/Diff Exp = DESeq2
Differential Gene Expression
• Is the difference in gene expression statistically significant ?
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Gene ID
CONDITION A CONDITION B Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3
001 132 151 98 1239 849 1563 002 2063 1825 1911 2107 2046 2031 003 12585 12158 12858 320 362 316
FOLD CHANGE
P-VAL
-3.3 1.9E-46 -0.12 0.51 5.2 1E-224
Table of raw counts
Are replicates correlated?
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Biological Replicate set -highlighted in white box
Outlier
Rep1 Rep2 Rep3
Condition 1
High Low
Diagonal- Replicate vs itself
Condition 1
Rep1 Rep2 Rep3
Condition 3
Condition 2 Rep1 Rep2 Rep3 Rep1 Rep2 Rep3
Pearson Correlation
Condition 2 Condition 3
RNA-Seq Workflow
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RNA Samples
RNA Library Construction
Data QC
Genome Annotation
Differential Expression
Data to User
Small RNA Analysis
Small RNA Analysis – miRDeep2
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Total RNA
Provisional ID : chromosome:AGPv2:7:1:176764762:1_38876Score total : -6.7Score for star read(s) : -1.3Score for read counts : 0Score for mfe : -3.2Score for randfold : -2.2Score for cons. seed : Total read count : 26Mature read count : 26Loop read count : 0Star read count : 0
5'uc g g
a c c a g g cuuca a u
c cc u u u a a cua g c
gu c u
g c a u au a
uaugugc
uucucuaau
cagcuguucaag
caauu
ugccucugggu
3'
freq.
length
1
0.75
0.5
0.25
01
Mature
22 70
Star
86
ggguguaccuguuggugaucucggaccaggcuucaaucccuuuaacuagcgucugcauauauaugugcuucucuaaucagcuguucaagcaauuugccucuggguaagcc -3'5'- exp
ggguguaccuguuggugaucucggaccaggcuucaaucccuuuaacuagcgucugcauauauaugugcuucucuaaucagcuguucaagcaauuugccucuggguaagcc known
(((....)))...(((...((((((...(((...(((..(((.(((.(((....(((((....)))))...........)))))).)))..))).)))))))))...))) reads mm sample
.................aucucggaccaggcuucaUucccu..................................................................... 1 1 seq
..................ucucggaccaggcuucaGucc....................................................................... 1 1 seq
....................ucggaccaggcuucaaucc....................................................................... 1 0 seq
....................ucggaccaggcuucaauccc...................................................................... 1 0 seq
....................ucggaccaggcuucaaucccC..................................................................... 6 1 seq
....................ucggaAcaggcuucaaucccu..................................................................... 1 1 seq
....................ucggaccaggcuucaUucccu..................................................................... 3 1 seq
....................ucggaccaggcuucaaucccu..................................................................... 13 0 seq
Novel miRNA miRNA expression Read Lengths
Small RNA Library Prep Sequencing
Example Symbiont Project
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Goal: Identify symbiotic gene expression effects Known: Fungal infection increase plant growth Design: Plant / Fungi grown in isolation and in contact
1
4
16
64
256
1024
4096
16384
1 8 64 512 4096
Log2 fold change (Isolation)
Log2
fold
cha
nge
(Con
tact
)
Plant
1
4
16
64
256
1024
4096
16384
1 8 64 512 4096
Log2 fold change (Isolation)
Log2
fold
cha
nge
(Con
tact
) Fungi
Plant in Isolation
Plant in Contact
q
Fungi in Isolation
RNA-Seq Workflow
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RNA Samples
RNA Library Construction
Data QC
Genome Annotation
Differential Expression
Data to User
Small RNA Analysis
RNA-SEQ Data Provided Through JGI Genome Portals
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FY 2015 RNA Projects
734 Fungal Samples
146 Microbial Samples 466 Metatranscriptome Projects
2754 Plant Samples
http://genome.jgi.doe.gov
Who’s Who ?
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QC
Bryce Foster
Analysis
Bill Andreopoulos Erika Lindquist Brian Foster Anna Lipzen
Lead Community MT Fungal Microbial
Sequencing Technologies
Chris Daum Rita Kuo Yuko Yoshinaga
Project Management
Kerrie Barry Tijana Galvina del Rio Christa Pennacchio Vivian Ng