Tag profiling is dead...

October 2009

Claudia Voelckel Patrick Biggs

...long live mRNA-Seq!

Expression Studies in the New Zealand Flora

Species diversification & local adaptation

Hybridization & polyploidy

Biological processes that differ between species and populations

Adaptive gene sets

We are interested in:

Ranunculus Ourisia Hebe

Pachycladon Nothofagus Totara

3

DNA chip

with gene probesAAAAAA3’TTTTTT5’

TTTTTT5’green-labeled cDNA

AAAAAA3’TTTTTT5’

TTTTTT5’ red-labeled cDNA

Expression Studies the Familiar Way: Microarrays

Sample 1

AAAAAA3’mRNA

Sample 2

AAAAAA3’ mRNA

DATA ANALYSISintensity 1intensity 2

Expression ratio: log

4

Expression Studies Revolutionized: Tag Profiling

12

21

11

count 1count 2

log

STATISTICAL ANALYSIS

Solexa Genome Analyzer

Sample 1 mRNA

AAA3’AAA3’

AAA3’AAA3’

Sample 2mRNAAAA3’

AAA3’AAA3’

AAA3’

18 bp tag library

AAA3’

AAA3’

AAA3’

AAA3’

18 bp tag library

AAA3’

AAA3’

AAA3’

AAA3’

Sample 1

Sample 2

Reference

TAG MAPPING

Advantages & Challenges of Tag Profiling

open to any organism (with a reference transcriptome)

any expressed transcript detectable (1 copy/cell)

less RNA needed (tag profiling = 1µg, microarrays = 100 µg)

minor data normalization, cross-species comparisons easier

Advantages

Challenges

mapping 18 bp tags (sequence differences Pachycladon/Arabidopsis)

counting tags per gene (noise, location, abundance)

statistical analysis of differential expression (proportion data)

Tag Profiling Guinea Pig: Previous Microarray Study

Habitat Rosette

Flowering Fruiting

Habitat Rosette

Flowering Fruiting

Pachycladon fastigiata Pachycladon enysiivs.

Voelckel et al. 2008, Molecular Ecology, 17: 4740–4753Comparative gene expression study using Arabidopsis microarrays

P. enysiiP. fastigiata

Prob

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ty o

f diff

eren

tial e

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dds r

atio)

Magnitude of differential expression (log fold change)

ESM1 ESP

Arabidopsis microarray (20,468 genes)

310 genes (1.5%) up in P. fastigiata 324 genes (1.6%) up in P. enysii

up-regulation of ESP and ESM1 predict P. fastigiata to produce isothiocyanates and P. enysii to produce nitriles

prediction confirmed by HPLC

role for herbivory in species diversification?

Microarray Study Results

Tag Profiling Results

8 data sets from different mapping strategies (ELAND, MySQL) each analyzed with different normalization parameters (R, edgeR) results vary!

Example:

data set 2: 17423 A. thaliana loci noise filter 10 count most abundant tag per gene

analyzed with tagwise normalization -log2(1.5) < log fold ratio < log2 (1.5)

2654 genes (15.2%) up in P. fastigiata 1857 genes (10.7%) up in P. enysii

P. enysiiP. fastigiata

Microarrays (MA) vs. Tag Profiling (TP)

more differentially expressed genes in TP (10.7-15.2% ) than with MA (1.5-1.6% )

MA: 20,468 genes

310 up in PF324 up in PE

TP: 17,423 genes

2654 up in PF1857 up in PE

Locus lfc MA lfc TPAT3G14210, ESM1 -4.6 -35.0

Locus lfc MA lfc TPAT1G54040, ESP 6.3 7.0AT2G14750, APK 0.7 0.9AT2G43100, MAM-I 0.9 1.9AT5G14200, MAM-D 0.7 1.1AT5G23010, MAM1 0.8 1.4

biological inferences from both studies identical

PF

MA TP41269 2613

PE

50274 1807MA TP

13.2% (PF) and 15.4 % (PE) of MA results confirmed by TP results

“...not a popular product, too expensive, tricky chemistry.. instead use:

mRNA-Seq!”

Tag Profiling is dead, long live mRNA-Seq!

2 Oct 09: “Illumina is discontinuing the support of Tag Profiling and will no longer be manufacturing the reagent kits for this application.”

One year later: Tag profiling works for a non-model plant with a distant reference transcriptome! Let’s do more experiments!

11

Expression Studies Revisited: mRNA-Seq

Sample 1

AAA3’AAA3’

AAA3’AAA3’

Sample 2mRNA mRNA

Solexa Genome Analyzer

AAA3’

AAA3’AAA3’

AAA3’

cDNA library cDNA library

Sample 1

Sample 2

ReferenceREAD MAPPING

12

21

11

count 1count 2

log

STATISTICAL ANALYSISgene length

new mapping strategies needed

different statistical treatment required

hardly any R packages available yet

Advantages & Challenges of mRNA-Seq

Advantages

Challenges

whole transcriptome coverage

longer reads reduce mapping noise and unmapped reads

multiplex-compatible

adequate coverage (too high with tag profiling)

additional benefits: EST libraries, SNPs

disentangling expression of allopolyloid copies may be easier

Ranunculus Ourisia Pachycladon

Experiences with mRNA-Seq

Tuatara

mRNA-Seq runs (75bp paired end) so far:

await assembly and analysis

EST data base built for

P. fastigiata

analysis in progress

Straight from the Pachycladon EST library:

Evidence for allopolyploid copies (e.g. glucosinolate hydrolysis gene)

THANKS TO:

Genome Service

Patrick Biggs

Lorraine Berry

Lesley Collins, Maurice Collins, Pete Lockhart

Helene Kretzmer

Marsden

Alexander von Humboldt Foundation