rna-seq at jgiusermeeting.jgi.doe.gov/wp-content/uploads/sites/2/2016/...2016/04/06  · rna-seq...

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