Download tileMEseq0905

Notes and
statistics on
base level
expression
Don Gilbert
May 2009
Biology Dept., Indiana University
[email protected]
wfleabase.org/docs/tileMEseq0905.pdf
2007: Tile expression
DrosMel tiled by Affymetrix, finds new genes (blue) and
known (orange)
.
wfleabase.org/docs/tileMEseq0905.pdf
Precision improves ’06-’09
Measuring expression over gene structures,
Nimblegen (08) has higher precision than Affy (06/07)
RNA-Seq (09) has higher precision than Nimblegen
.
wfleabase.org/docs/tileMEseq0905.pdf
…
microarray statistics
for base level expression?
wfleabase.org/docs/tileMEseq0905.pdf
Gene or Base expression?
•
Base-level expression (tiles, rna-seq) calculate like
gene differential expression (DE)
•
•
Combine for tiles over gene
•
•
•
•
Per tile, per RNA-seq contig or per base: treatment - control
Independent (technically) observations, but biologically related
Increase DF, Power with longer gene
How to combine?
•
As independent replicates: gene > (tiles, technical, bio replicates)?
•
As nested block: gene > tiles > replicates ?
•
As gene average: gene = mean(tiles) > replicates ?
Compare with gene-level stats
…
wfleabase.org/docs/tileMEseq0905.pdf
Gene or Base expression?
Base level tests find expression better than gene average
Base level sensitivity= 42%, Gene level sensitivity= 38% Both have specificity= 37%
Sensitivity = 1 - false rejection; Specificity = 1 - false discovery
wfleabase.org/docs/tileMEseq0905.pdf
Gene or Base expression?
DE is consistent over gene span though expression Ave
changes; gene-level measure can miss this.
Expression over gene span, treatment(red) vs control(green) with 3 replicates
wfleabase.org/docs/tileMEseq0905.pdf
…
gene structures & expression
wfleabase.org/docs/tileMEseq0905.pdf
Sequence normalizing?
Idea is to remove sequence (GC) effects on probe hyb. score
QuickTime™ and a
decompressor
are needed to see this picture.
TileScope; Royce TE, Rozowsky JS, and Gerstein, MB. (2007). Assessing the need for
sequence-based normalization in tiling microarray experiments. Bioinformatics, 23, 988-997.
wfleabase.org/docs/tileMEseq0905.pdf
Sequence normalizing?
Sequence-normalizing also removes Exon/Intron signal !
QuickTime™ and a
decompressor
are needed to see this picture.
Don’t use it (TileScope’s quantilenorm)
.. or other sequence adjustments of expression,
unless gene structure signals are included.
wfleabase.org/docs/tileMEseq0905.pdf
QuickTime™ and a
decompressor
are needed to see this picture.
Intron-Exon Detection
Nimblegen and Solexa tile/base expression detects gene structure, on average, fairly well.
wfleabase.org/docs/tileMEseq0905.pdf
Intron-Exon Update
Newest
RNA-Seq
finds
intron/exon
very well
(Stranded RNASeq, modEncode
Gingeras lab,
March 2009 )
wfleabase.org/docs/tileMEseq0905.pdf
Differential expression
Gene end (3’) has more expression, but
Example
genes
exons
introns
QuickTime™ and a
decompressor
are needed to see this picture.
constant differential over gene span, on average.
Green is treatment, red control. Line style shows 3 replicates of Daphnia tiled expression.
wfleabase.org/docs/tileMEseq0905.pdf
Diff. Expr. distributions
Introns show a Pred
null DE
distribution,
genes and
TAR regions Metal
are wider.
Genes
Use introns as
baseline for
Sex
statistics?
wfleabase.org/docs/tileMEseq0905.pdf
Introns
TARs
…
multiple testing corrections
wfleabase.org/docs/tileMEseq0905.pdf
Multiple statistic tests
• Problem: perform 20,000 tests and p-values hit laws of
chance. Pr = 0.05 can happen 1,000 times by chance (false
discovery, FDR).
• DrosMel Affy line t-tests: 2,284,383 / 5,395,023 = 0.42 Sig
• Bonferroni: conservative = 0.03 Sig
• Benjamini & Hochberg: p.adjust(p,’BH’) = 0.35 Sig
• qvalue(p) : distribution based = 0.41 Sig
Storey, JD and R Tibshirani, 2003. Statistical significance for genomewide studies. PNAS 100:94409445
• SAM permutation qvalue
• However, p.adjust meant for 100’s of tests, not Millions
• Drosmel modEncode case: 1900 pairwise Affy cell line (62 cells) DE
comparisons x 14,000 genes = 26,600,000 t-tests
wfleabase.org/docs/tileMEseq0905.pdf
Multiple DE tests : Daphnia
Sex
P<0.05 6733
%P
28
%BH
19
%Qvalue
21
max P|Q 1e-2
Predate
832
3
0
0
1e-4
Metals
2502
10
0
0
1e-4
• Much different corrections for experiments on same genes
• Daphnia DE: 3 expt.s (trt - con), 25000 genes, 3 replicates
• Predate, Metal genes have low expression, important to detect
wfleabase.org/docs/tileMEseq0905.pdf
Multiple statistic tests
• “Statisticians have turned p-value corrections into
an industry, but they are really more of a band-aid
than a solution”*
• What about false rejection (FRR; type II error)?
• Balance errors, false rejection maybe more important
• Solution #1: test fewer, directed hypotheses
• Solution #2: measure error rate on knowns, eg.
prediction of “known” genes
• Solution #3: known null hypothesis, eg. introns
*http://www.bioconductor.org/workshops/2009/SeattleApr09/DiffExpr/
wfleabase.org/docs/tileMEseq0905.pdf
1900 pairwise Affy cell line DE comparisons x 14,000 genes = 26,600,000 t-tests
QuickTime™ and a
decompressor
are needed to see this picture.
wfleabase.org/docs/tileMEseq0905.pdf
Hypotheses of interest are fewer: ~100s cells x 14,000 genes ~ 2 Million tests
QuickTime™ and a
decompressor
are needed to see this picture.
wfleabase.org/docs/tileMEseq0905.pdf
Summary
1. Base-level expression (tiles, rna-seq) measures
gene expression better
•
Balances sensitivity (false rejection) with specificity (false discovery)
2. Base-level expression measures gene structures
well
•
On average, and precision is improving for individual genes.
3. Multiple test corrections are needed but
problematic
•
•
False discovery corrections for millions of tests leads to false
rejections.
Determine empirical error rates where possible
wfleabase.org/docs/tileMEseq0905.pdf
End note
Summary pages
wfleabase.org/genome-summaries/tile-expression/
insects.eugenes.org/species/data/dmel5/modencode/
Genome expression maps
insects.eugenes.org:8091/gbrowse/cgi-bin/gbrowse/drosmelme/
•
expression in 52 cell lines (affy) and more precise solexa & nimblegen for
a few cell lines
insects.eugenes.org:8091/gbrowse/cgi-bin/gbrowse/daphnia_pulex8/
•
expression among 4 treatment groups (sex, metal stress, biotic predator);
nimblegen
wfleabase.org/docs/tileMEseq0905.pdf