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iSelect, CSPro, and GenomeStudio are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.

Welcome!

Abizar Lakdawalla, PhD

alakdawalla@illumina.com

• Pervasive transcription

• Antisense transcription

• Non-coding RNA

• Promoter associated RNA

• Micro-RNA/mRNA targets

• Enhancer RNAs

• Gene fusions

Transcriptome sequencing

Goodbye!

Arrays

22 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Human Genome circa 2010

33 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

44 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

RNA families

RNA

Coding

PolyA

mRNA

Non-PolyA

mRNA

Non-coding

Structural

DNA associated

Replisome

DNA Repair

Telomeric

DNA methylation

(piRNA)

RNA associated

Ribosome associated

rRNA

Regulatory

Micro RNA

TSS associated

Anti-sense

Enhancer RNA

55 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Genome Analyzer Transcriptional Analysis

RNA from mitochondria

RNA from nucleus

RNA from cytoplasm

Sub-fractionate the RNA

• Poly-A RNA

• RNA without rRNA

• RNA without …Sequence the whole RNA

Tag Sequence

(a snippet)

66 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Tag Sequencing

AAAAAmRNA / Total

RNA

Sequencing Adapter

Ligation

GATC AAAAATTTTT-CTAG

MmeI

AAAAATTTTT-

1st and 2nd Strand

cDNA Synthesis

Restriction Enzyme

Digestion

GATC AAAAATTTTT-

77 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

2nd Primer Addition

via PCR

Seq. PrimerPrimer Tag Primer

Digital Tag mRNA Sample Preparation

GATC AAAAATTTTT-CTAG

MmeI

NN

MmeI Digestion

Ligation of

1st Primer

NNNN

88 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Gene Expression

250 samples per run (HiSeq 2000)

99 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

RNA-Seq: Method

AAAA AAAAAAA AAAAAA

Illumina Sequencing

cDNAsample

Genome position

Re

lati

ve

# o

f re

ad

s

1010 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

We can calculate the expression

ratio of any two samples at every

region across the entire genome

1111 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

1212 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

mRNA-Seq Data is Information Rich

mRNA Expression Profiling

Alternative Splicing Analysis

Analysis of expressed SNPs and mutations

Analysis of Allelic-specific Expression

Chimeric Transcript Discovery

Gene Discovery and Annotation

1313 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Base Position 51,165,353 51,166,593 51,167,833 51,169,073 51,170,313 51,171,553 51,172,793 51,174,033 51,175,273 51,176,513

Cytogenetic Band q13.13

Sequence (+) Sequence is too dense for display.

BCnormal_619K…

KRT6A

Base Position 151,798,793 151,800,683 151,802,573 151,804,463 151,806,353 151,808,243 151,810,133 151,812,023 151,813,913

Cytogenetic Band q21.3

Sequence (+) Sequence is too dense for display.

BCnormal_619K…

S100A2

Base Position 56,150,388 56,152,598 56,154,808 56,157,018 56,159,228 56,161,438 56,163,648 56,165,858 56,168,068

Cytogenetic Band q13.33

Sequence (+) Sequence is too dense for display.

BCnormal_619K…

KLK6

KLK6

normal

tumor

KRT6A S100A2 KLK6

1414 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Tissue-specific genes

Base Position 125,121,475 125,121,905 125,122,335 125,122,765 125,123,195 125,123,625 125,124,055 125,124,485 125,124,915

Cytogenetic Band q24.2

Sequence (+) Sequence is too dense for display.

adipose

PATE1

PATE1 Gene

Prostate And Testis Expressed 1

1515 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Read Counts for All RefSeq Genes

1616 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

RNA-seq and Microarray Compared

Expression levels are shown, as measured by RNA-Seq and tiling

arrays for Saccharomyces cerevisiae cells. Agree for genes with

medium levels of expression, but correlation is very low for genes

with either low or high expression levels.

1717 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Dynamic Range

Accuracy and

Sensitivity

Performance of RNA sequencing is superior to other gene

expression profiling methods

1818 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

mRNA-Seq

1919 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Transcribed region of PMPCB gene is clearly longer than RefSeq annotation

Overlapping of PMPCB and DNAJC2 genes on two

strands

2020 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

30%

2121 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

cSNP AnalysisHomozygous

Heterozygous

2222 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Differential Allelic Expression

2323 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Read Counts for All RefSeq Exons

2424 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Transcriptome isoforms –

2525 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Making Sense

“… studying transcription for a long

time and never seen this kind of

transcription before … but we also

see a polymerase that appears to be

pointing in the wrong direction.” -

Seila/Sharp, MIT

“The gene, in other words, is in an identity crisis.”

2626 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Gene Fusions

2727 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

2828 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Foxa2 Binds in Unknown Regions

… two remote peaks in regions containing no known genes. The first peak (unknown

#4) lies close to a mouse EST, while the second peak (unknown #5) overlaps and is

close to highly conserved sequence regions.

2929 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

3030 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

More to small RNA than known microRNAs

miRNA FragmentPotential

Novel Content

rRNA, tRNA, adaptor contamination

Standard Protocol: <3%

sRNA v1.5 30 nt band: 6%

sRNA v1.5 22 nt band: 8%

Non-coding RNA Fragment

3131 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

More to small RNAs ...

3232 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Identification of micro-RNA targets

Henderson, 2009

3333 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Argonaute HITS-CLIP

decodes microRNA-

mRNA interaction maps.

Chi SW, Zang JB, Mele A,

Darnell RB. Nature (2009)

460: 479-86.

Covalently crosslink native

argonaute protein–RNA

gives you Ago–miRNA and

Ago–mRNA binding sites.

Ago-mRNA tags to genome

3434 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Ribo-Seq.

RNA engaged in translation

Ribo-Seq

RNA-Seq

3535 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

RNA Normalization

3636 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Genome Analyzer Transcriptional Analysis

RNA from mitochondria

RNA from nucleus

RNA from cytoplasm

Sub-fractionate the RNA

• Poly-A RNA

• RNA without rRNA

• RNA without …Sequence the whole RNA

Tag Sequence

(a snippet)

3737 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Cot Curves and cDNA Normalization

rRNA

mtRNA

mRNA

3838 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Double strand nuclease normalization

mRNA-Seq

Purified Total RNA

Poly-A Selection

RNA Fragmentation

cDNA Synthesis*

Adapter Ligation & PCR

Total RNA-Seq

Purified Total RNA

RNA Fragmentation

cDNA Synthesis*

Adapter Ligation & PCR

DSN Normalization

3939 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

DSN Library Normalization Process Overview

From Evrogen.com

Library is melted at 98ºC

Library is hybridized at 68ºC

DSN Cleavage

PCR to enrich library

Grow Clusters and Sequence

4040 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Total RNA-Seq Method Overview

RNA from any organism – bacteria, plants, animals

Less than 100 nanograms of total RNA

Full-length cDNA coverage of all RNA molecules

Works with low quality RNA – even FFPE preparations

Normalization protocol can be used with most RNA-Seq

protocols

4141 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Comparison of Output from Different Protocols

4242 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Many ncRNAs are often transcribed from the same strand as mRNA

Poly-A + DSN

Total RNA + DSN

4343 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Total RNA + DSN

Poly-A + DSN

Many non-coding RNAs are seen with Total RNA-Seq

4444 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

DSN Normalization has little effect on most mRNAs

Poly-A mRNA (no DSN)

Po

ly-A

m

RN

A

(+ D

SN

)

4545 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Plot shows gene level count data

for all RefSeq Genes

Total RNA vs. Poly-A mRNA

Many non-coding RNAs are observed with

Total RNA-Seq MethodTo

tal

RN

A

Poly-A mRNA

4646 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

-10 -5 0 5 10

-10

-50

51

0

Log2 sequencing count ratio (brain vs UHR)

Lo

g2

qP

CR

ra

tio

(b

rain

vs U

HR

)

R = 0.947

Slope = 1.01

Fold

Change for

qR

T-P

CR

:

Bra

in/U

HR

Fold Change for Total RNA-SEQ:

Brain/UHR

Key Variables:

RNA is degraded

No Poly-A Selection

+ DSN Normalization

0.1 µg input total RNA

Fold Change of Total RNA-Seq vs. qRT-PCR

4747 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

RNA-Seq in FFPE

4848 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

RNA-Seq Protocol Variations

mRNA-Seq

Purified Total RNA

Poly-A Selection

RNA Fragmentation

cDNA Synthesis*

Adapter Ligation & PCR

Total RNA-Seq

Purified Total RNA

RNA Fragmentation

cDNA Synthesis*

Adapter Ligation & PCR

Normalization

Total RNA-Seq

Total RNA from FFPE

cDNA Synthesis*

Adapter Ligation & PCR

Normalization

4949 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Zoom in

Collaboration with Chuck Perou - UNC

Total RNA-Seq protocol on FFPE Samples

Two Cancer Samples– Her2 and Basal breast cancer sub-types

Two types of RNA preparations– Total RNA from fresh/frozen tissue and highly degraded FFPE RNA

RIN of the fresh samples are 9.3 and 8.3

Fresh Samples

FFPE Samples

5050 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

10-2

10-1

100

101

102

103

104

s5_csv1.7_c Counts

s1_csv1.7_c Counts vs s5_csv1.7_c Counts

10-2

10-1

100

101

102

103

104

s1

_c

sv1

.7_

c C

ou

nts

R2 = 0.89

FFPE Total RNA-Seq vs. Fresh Total RNA-Seq

Pair-wise Comparison of FFPE

5151 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

MUCL1(mucin-like 1 gene)

FFPE - Basal

FFPE - Her2

Fresh mRNA – Basal

Fresh mRNA - Her2

Fresh Total RNA - Basal

Fresh Total RNA – Her2

Fresh Total RNA - Basal

Fresh Total RNA – Her2

Replicate 1

Replicate 2

5252 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Whole Transcriptome Studies on HiSeq 2000 System

Format

PF

readsAverage Error Rates

(PhiX spike-ins) Yield

Individual human tissues 2 X 50 2.55 B 0.22%, 0.44% >120 Gb in <5 days

Individual human tissues 1 X 75 1.27 B 0.37%, 0.52% >95 Gb in <4 days

Total human transcriptome 1 X 100 1.19 B 0.67%, 0.81% >115 Gb in <5 days

Body Map 2.0 ProjectAdrenal, Adipose, Brain, Breast, Colon, Heart,

Kidney, Liver, Lung, Lymph Node, Ovary, Prostate,

Skeletal Muscle, Testis, Thyroid, White Blood Cells

INDIVIDUAL HUMAN TISSUES

16 tissues, 1 lane per tissue

Standard Poly-A mRNA-Seq Library Preps

TOTAL HUMAN TRANSCRIPTOME

Equal Mixture of 16 Human Tissues

Strand-specific mRNA-Seq

SUMMARY OF RUN METRICS

5353 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

PCR-free

5454 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

No PCR

No PCR

PCR

PCR

GAPDH

5555 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

No PCR

No PCR

PCR

PCR

APOB

5656 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

No PCR vs PCR (15 cycles)

100

101

102

103

104

105

HCT10738 Counts (Raw)

HCT10736 Counts (Raw) vs HCT10738 Counts (Raw)

100

101

102

103

104

105

HC

T1

07

36

Co

un

ts (

Ra

w)

R2=0.9637

5757 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Thank you

Summary

RNA-Seq = more publications!

DATA

ANALYSES

5858 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Samples/flow cell and cost/sample (all consum.)

GAIIe GAIIx HiScanSQ HiSeq 2000

DGE (25bp, 2

million)

60

€ 230

125

€ 205

125

€ 204250

€ 192

RNA-Seq (75x2, 20

million)

6

€ 1585

13

€ 898

13

€ 83525

€ 522

ChIP-Seq (TF) 125

€ 252

250

€ 231

250

€ 229500

€ 219

Virus/BAC clones

(100x2, 30x)

2,770

€ 209

5,555

€ 207

5,555

€ 20711,111

€ 206

Bact./GWAS

(100x2, 30x)

277

€ 242

556

€ 224

556

€ 2221,111

€ 214

Hu exome (100x2,

30x)

28

€ 568

56

€ 387

56

€ 368111

€ 287

Hu genome (100x2,

30x)

0.28

€ 36,444

0.56

€ 18,325

0.56

€ 16,4361.11

€ 8,321€ 5,616

@300Gb/run

5959 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

1270 genes respond to beta-estradiol, includes several direct

targets of Eralpha, organized in a gene regulation cascade,

stemming from ligand-activated receptor and reaching a large

number of downstream targets via AP-2gamma, B-cell activating

transcription factor, E2F1 and 2, E74-like factor 3, GTF2IRD1, hairy

and enhancer of split homologue-1, MYB, SMAD3, RARalpha, and

RXRalpha transcription factors.

MicroRNAs integral components of this gene regulation network;

miR-107, miR-424, miR-570, miR-618, and miR-760 are regulated

by 17beta-estradiol along with other microRNAs that can target a

significant number of transcripts belonging to one or more estrogen-

responsive gene clusters.

Estrogen receptor alpha controls a gene network in luminal-like breast cancer

cells comprising multiple transcription factors and microRNAs. Cicatiello L …

Am J Pathol (2010) 176: 2113-30.

6060 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Genomic DNA

X X

Restriction Enzyme Digestion

Size Selection (200+25bp)

Bisulfite Treatment

Sequence Ends of Selected Fragments

Data Analysis

200 +/- 25 bp

Reduced Representation Bisulfite Seq

6161 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

BC Cell Line RRBS Data - MMEL1 Gene

MCF7

ZR-75-1

T47D

BT474

MDA-MB-231

MDA-MB-468

BT20

MCF10A

6262 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

BC Cell Line RRBS Data - CD247 Gene

MCF7

ZR-75-1

T47D

BT474

MDA-MB-231

MDA-MB-468

BT20

MCF10A

6363 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

BC Cell Line RRBS Data - CDH13 Gene

MCF7

ZR-75-1

T47D

BT474

MDA-MB-231

MDA-MB-468

BT20

MCF10A

6464 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Correlation of RRBS and mRNA-Seq Data for PRKD1 Gene

chr14:29,027,620-29,554,515 PRKD1 gene methylation for 5’ CpG island region been

dramatically changed and anti-correlates well with mRNA levels

MCF7

ZR-75-1

T47D

BT474

MDA-MB-231

MDA-MB-468

BT20

MCF10A

109

37

0

283

0

0

0

418

mRNA levels

BT474

mRNA levels

6565 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

LungBreast

CDH13 (H-cadherin): increased methylation in lung tumor

3% ave

47% ave

4% ave

10% ave

Study both individual CpG sites and CpG Islands

6666 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

CpG island sumMeth value in twins

0

5000

10000

15000

20000

25000

30000

35000

0 5000 10000 15000 20000 25000 30000 35000

twin1

twin

2Tw

in 1

01

Twin 001

CpG island sumMethyl value in breast cancer

0

5000

10000

15000

20000

25000

30000

35000

0 5000 10000 15000 20000 25000 30000 35000

normal breast

be

as

t tu

mo

r B

Bre

ast

Can

cer

Normal Breast

CpG island sumMeth value in lung cancer

0

5000

10000

15000

20000

25000

30000

0 5000 10000 15000 20000 25000 30000

lung normal

lun

g t

um

or

B

Normal Lung

Lun

g C

ance

r

Comparison of Methylation Summarized for ~ 25K CpG Islands

6767 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Whole Genome Bisulfite SequencingReduced Representation Bisulfite Sequencing

6868 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

CpG Island Methylation Changes & Pathways

What are in these pathways that change?

Top 3 Pathways by Enrichment

cAMP-mediated Signaling

(p = 2.09e-8)

G-Protein Coupled Receptor Signaling

(p = 4.17e-7)

Axonal Guidance Signaling

(p = 1.18e-6)

Cell Death

Related

Pathways

Implication: increased methylation of cell death related

pathways could promote cell growth and cancer progression

6969 Revolution – RNA-Seq – PCR-free – Ribo-Seq – CLIP-Seq – Normalization - FFPE

Experimental Data Available

RNA-Seq

50 bp paired-end standard mRNA-Seq

100 bp single-read directional mRNA-Seq

DNA Methylation - RRBS

CNV- low pass genomic DNA sequencing

Small RNA Analysis