rna-seq: sequencing the transcriptome...sesb sesb cg15211 cg15211 cg15211 cg15211 ant2 ant2 sesb...
Post on 22-Jan-2021
22 Views
Preview:
TRANSCRIPT
RNA-Seq: Sequencing the Transcriptome
Kasper Daniel HansenDivision of Biostatistics, UC Berkeley
Using Bioconductor for ChIP-Seq experimentsFHCRC, Seattle 12th-14th of November 2008
RNA-Seq: Comparison with Microarrays
Potential for surveying the entire transcriptome, including novel, un-annotated regions.
Potential for determining gene structure and isoform level expression using reads mapping to splice junctions.
Potential for making better presence/absence calls on regions.
Con: the assay is dependent on sequencing effort, low expressed regions will be missed.
Protocol
The current standard protocol for RNA-Seq is
Extraction of RNA, polyA purification Fragmentation of RNA RT of RNA to cDNA Ligation of adapters Size selection ~ 200bp (perhaps ~300bp) PCR amplification (15 rounds) Injection into flowcell
This produces reads from polyadenylated RNA without strand information.
Attempts are being made to make the assay strand specific and to assay total RNA as well.
Data from D. melanogaster:chrX
Conservation
d_simulansd_sechelliad_yakubad_erectad_ananassaed_pseudoobscurad_persimilisd_willistonid_virilisd_mojavensisd_grimshawia_gambiaea_melliferat_castaneum
1067300010673500106740001067450010675000106755001067600010676500106770001067750010678000106785001067900010679500106800001068050010681000
CG8144_6lane_0MM
S2_DRSC_6_lanes
FlyBase Protein-Coding Genes
RefSeq Genes
12 Flies, Mosquito, Honeybee, Beetle Multiz Alignments & phastCons Scores
Repeating Elements by RepeatMasker
CG15211CG15211CG15211CG15211
Ant2Ant2
sesBsesBsesBsesB
CG15211CG15211CG15211CG15211
Ant2Ant2
sesBsesBsesBsesB
_ 1000
_ 0
_ 1000
_ 0
ps RNAi
S2 Untreated
Splice JunctionReads
GenomicReads
Splice JunctionReads
GenomicReads
Image from Brenton Gravely
Base effect - single sample
14.1m reads
369860 369880 369900 369920 369940
0.0
00.0
10.0
20.0
30.0
4
gene: YLR110C
position
pro
port
ion o
f to
tal re
ads in r
egio
n
Base effect - multiple samples
369860 369880 369900 369920 369940
0.0
00.0
10.0
20.0
30.0
4
gene: YLR110C
position
pro
port
ion o
f to
tal re
ads in r
egio
n
wt
rrp
ski
Base effect - different study (and prep)
369860 369880 369900 369920 369940
0.0
00.0
10.0
20.0
30.0
4
gene: YLR110C
position
pro
port
ion o
f to
tal re
ads in r
egio
n
WT
Nagalakshmi
Base effect - different prep
369860 369880 369900 369920 369940
0.0
00.0
10.0
20.0
30.0
4
gene: YLR110C
position
pro
port
ion o
f to
tal re
ads in r
egio
n
original
ribominus
Base effect - different aligners50 100 150
050
100
150
MAQ
position
counts
UHR(MAQ)
UHR(ELAND)
50 100 150
0.00
0.02
0.04
MAQ
position
counts
MAQ and ELAND, Human data
Base effect - conclusions
Reproducible base effect - like probe affinities in microarrays.
Seems to be prep dependent.
Creates issues for comparing differentregions in the genome.
Less of an issue for comparing thesame region across samples?
369860 369880 369900 369920 369940
0.0
00
.01
0.0
20
.03
0.0
4
gene: YLR110C
position
pro
po
rtio
n o
f to
tal re
ad
s in
re
gio
n
?
Mapping reads to the transcriptome
Transcriptome
2^Genome
Reads
Genome
Illustration from Lior Patcher
Well established
Mapping transcripts
Genome
Transcript
Length in genome space
paired-end reads
Junction reads:chrX
Conservation
d_simulansd_sechelliad_yakubad_erectad_ananassaed_pseudoobscurad_persimilisd_willistonid_virilisd_mojavensisd_grimshawia_gambiaea_melliferat_castaneum
1067300010673500106740001067450010675000106755001067600010676500106770001067750010678000106785001067900010679500106800001068050010681000
CG8144_6lane_0MM
S2_DRSC_6_lanes
FlyBase Protein-Coding Genes
RefSeq Genes
12 Flies, Mosquito, Honeybee, Beetle Multiz Alignments & phastCons Scores
Repeating Elements by RepeatMasker
CG15211CG15211CG15211CG15211
Ant2Ant2
sesBsesBsesBsesB
CG15211CG15211CG15211CG15211
Ant2Ant2
sesBsesBsesBsesB
_ 1000
_ 0
_ 1000
_ 0
ps RNAi
S2 Untreated
Splice JunctionReads
GenomicReads
Splice JunctionReads
GenomicReads
Image from Brenton Gravely
Junction reads, zoom
:chrX
Conservation
d_simulansd_sechelliad_yakubad_erectad_ananassaed_pseudoobscurad_persimilisd_willistonid_virilisd_mojavensisd_grimshawia_gambiaea_melliferat_castaneum
1067300010673500106740001067450010675000106755001067600010676500106770001067750010678000106785001067900010679500106800001068050010681000
CG8144_6lane_0MM
S2_DRSC_6_lanes
FlyBase Protein-Coding Genes
RefSeq Genes
12 Flies, Mosquito, Honeybee, Beetle Multiz Alignments & phastCons Scores
Repeating Elements by RepeatMasker
CG15211CG15211CG15211CG15211
Ant2Ant2
sesBsesBsesBsesB
CG15211CG15211CG15211CG15211
Ant2Ant2
sesBsesBsesBsesB
_ 1000
_ 0
_ 1000
_ 0
ps RNAi
S2 Untreated
Splice JunctionReads
GenomicReads
Splice JunctionReads
GenomicReads
Image from Brenton Gravely
Strategies for mapping to junctions
Map to known junctions.
Map to combination of known exons.
Map completely de-novo using canonical acceptor and donor sites. The combinatorics makes this an intimidating approach.
Map de-novo, but constrain the search to canonical acceptor and donor sites between and in transcribed region: transcript assembly. This is the approach taken by TopHat.
Paired-end data will make de-novo mapping a real possibility.
Mapping - conclusions
Mapping to transcript space is not easy.
But essential for really understanding alternative splicing.
Constructing all novel splice junctions based on canonical splice sites but only accepting splicing within genes (and small regions upstream/downstream of the gene) in D. Melanogaster yields 605,000,000 splice junctions.
0 5 10 15
02
04
06
08
01
00
Verified CDSdepth of 3
number of reads in millions
pe
rce
nt
bases s
eq
ue
nce
d
0 5 10 15
02
04
06
08
01
00
Dubious CDSdepth of 3
number of reads in millions
pe
rce
nt
bases s
eq
ue
nce
d
0 5 10 15
02
040
60
80
100
Uncharacterized CDSdepth of 3
number of reads in millions
pe
rce
nt
bases s
eq
ue
nce
d
0 5 10 15
02
04
06
08
01
00
Intronic Regionsdepth of 3
number of reads in millions
pe
rce
nt
ba
se
s s
eq
ue
nce
d
0 5 10 15
02
04
06
08
01
00
Background Regionsdepth of 3
number of reads in millions
pe
rce
nt
ba
se
s s
eq
ue
nce
d
wt
rrp
ski
xrn
Nagalakshmi
Detection in Cerevisiae
0 20 40 60 80 100
020
40
60
80
100
Intronic Regions
Percent introns detected
perc
ent genes d
ete
cte
d
wild type
!rrp6!lsm1!pat1
!ski2!ski8!rrp6
!xrn1!rrp6!lsm1!pat1
Nagalakshmi
0 20 40 60 80 1000
20
40
60
80
100
Background Regions
Percent background detected
perc
ent genes d
ete
cte
d
wild type
!rrp6!lsm1!pat1
!ski2!ski8!rrp6
!xrn1!rrp6!lsm1!pat1
Nagalakshmi
A. B.
Background: outside any transcribed feature, subtracted a boundary, subtracted any region detected as transcribed in recent studies
Detection in Drosophila
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
percent of intergenic regions detected
perc
ent of constitu
tive e
xons d
ete
cte
duntreated - 32.9m reads
brr2 - 15.0m reads
pasilla - 28.3m reads
1 read in 200bp, 10m reads
10 reads in 200bp, 10m reads
Replication
Sources of variation
Lane variation
Flowcell variation
Library prep variation
Biological variation
Poisson model
good fit
less good fit
Systematic differences
?: Is absolute quantification possible
Software
We have developed two R packages to help us
GenomeGraphs (Durinck, Bullard) : plots annotation and experimental data along a genome. Makes it easy to construct high quality images as well as to do data exploration. Available from Bioconductor.
Genominator (Bullard, Hansen) : provides support for managing, accessing and analyzing data oriented along a chromosome, together with annotation. Uses a SQLite backend. Works very well for unpaired reads mapped to the genome. Available from our home page.
R> summarizeByAnnotation(expData, annoData, fx)
Genome Graphs, example+
!
rrppolyA!
xrnTT!
rrp
xrn
wt
david!
snyder
nucleosome
miura!
conservation
YER015W YER016W
186500 187000 187500 188000 188500 189000
!
!
!!!
!
!
!!!
!!
!!!
!
!
!
!!!!
!
!!
!
!!!!
!
!!!
!
!!
!
!
!!!
!!!
!
!!!
!!
!
!!
!!!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!!!
!
!!
!!
!!
!
!!
!!
!
!!
!
!
!
!!!
!!!!
!
!
!
!
!
!
!
!
!!
!
!
!
!!!
!!
!
!
!
!!!
!!
!!
!
!!
!
!
!
!!
!
!!!
!!!!!
!!
!
!!
!
!
!!
!!
!
!!
!
!!
!
!
!
!!!
!!!!
!!!!
!!
!
!
!
!
!
!
!
!
!!
!!!
!
!
!
!!
!
!!
!
!
!
!!!
!!
!
!!
!
!
!!!!
!
!!
!
!!
!
!
!!!!!
!!
!!
!
!
!
!
!
!!
!
!!!
!!!
!!!!
!!!!!
!!!
!
!!
!
!
!!!!
!
!!!!!
!
!!
!!!
!
!
!
!
!!
!
!
!!
!!
!
!
!
!
!
!!!!
!
!!
!!!
!!
!
!
!!
!
!
!
!
!
!!!
!!!!
!!
!!!
!
!
!
!
!
!!
!
!!!!
!
!!
!!
!
!!
!
!
!
!!!!!!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!!
!
!!!
!!
!
!
!
!
!
!
!!!
!!
!
!
!!
!!
!!
!
!!
!
!
!
!!
!
!
!
!!!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!!!!
!
!
!!
!!
!!
!!
!
!
!
!
!
!
!!!!
!
!
!
!
!
!
!!
!!!
!!!
!
!
!
!!
!
!
!!
!
!
!!!!
!
!
!!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!!!
!!
!
!
!
!
!
!!
!!
!
!
!
!
!
!
!!
!!
!
!!
!!
!
!
!
!!
!
!
!
!
!
!!!
!!
!!
!
!
!!
!
!
!
!!
!
!
!!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!!
!
!!
!!
!
!!
!
!
!!!
!
!
!
!
!
!!!!
!!
!
!
!!!
!
!
!
!!
!
!
!!
!
!
!!
!!
!
!
!
!!
!
!
!
! !!
!!!
!3!2!1
0123
!!
!!!!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!
!!!!
!
!!!
!
!
!
!
!
!
!!!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!!
!
!!!!!!
!
!
!!!!
!!!
!
!
!!
!
!!
!
!
!
!!
!
!
!
!
!
!!
!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!
!!
!!
!!
!
!
!!
!
!!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!!
!
!!!
!
!!
!
!
!
!
!
!!
!
!
!
!!
!
!!!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!!
!
!
!
!!
!
!
!
!
!
!!
!!
!
!!!
!
!
!
!
!
!
!
!
!!
!
!
!
!!!
!
!
!!
!!
!
!
!!
!!
!
!
!
!!!
!
!!
!
!
!
!!
!
!
!
!!
!
!
!!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!!
!!
!
!
!!!!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!!!
!!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!
!!
!
!!
!
!
!
!
!
!
!
!
!!!!!!!
!!
!
!
!!
!!
!
!
!
!
!
!
!
!!
!
!
!
!
!!!!
!
!
!
!
!
!
!!
!
!
!
!!!
!
!
!
!
!!
!
!!
!
!
!!
!
!
!
!
!!
!
!
!
!
!!
!
!
!
!!
!!
!
!
!
!
!!!
!
!!!!
!
!
!
!
!
!
!
!
!
!
!!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!!
!!!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!3!2!1
0123
!!!!!!!!
!
!
!
!!! !
!
!!!!
!
!!
!
!!!
!
!!!
!
!!
!
!!!!!
!
!!!!!!!!!!!!!!!
!
!
!
!!! !!
!
!!
!
!
! !
!
!
!
!
!!!!
!
! !! !!!!!!!!!!!!
!
!
!!
!
!!!!!!!!!
!
!
!
!!
!! !!
!
!
!
!!!!!!!!!!
!
!!
!!
!
!!
!
!
!!
!!!!!
!
!
!!!!
!
!
!
!
!
!!!
!!
!!
!!!!!
!
!!
!
!!
!
!
!
!
!!!!
!
!
!!
!
!!
!
!!!
!
!!
!
!
!!!!!!!!!!!
!
!
!
! !!!
!
!!!!
!
!
!!!
!
!
!
!
!
!
!!!!!!!
!
!
!!
!
!
!
!!
!
!!!
!
!!!!
!
!!!
!
! !!!
!!
!!
!
!!
! !
!!!!!!!
!
!!!!!
!
!
!!!
!
!
!
!
!!
!!
!!!!!
!
!
!
!
!
!!
!
!
!
!!!!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!
!!!!!
!!
!!
!!
!
!
!
!!!!
!
!!!
!
!
!
!!
!
!
!
!
!
!!
!
!
!!!
!
!
!
!!
!
!
!
!
!
!!!!!!
!
!
!
!!!!
!!
!
!
!
!!
!!!
!
!
!
!
!!
!
!
!!
!
!!!
!
!
!!
!!
!!
!
!
!
!!
!
!
!
!
!
!
!!!
!
!
!
!
!!
!
!
!
!
!!!
!
!
!
!
!
!
!!!
!
!!!!!!!!
!!
!
!
!!!
!
!!
!
!!!!!
!!!!
!
!
!
!
!
!
!!
!
!!!!!!
!
!!
!
!!!!!!!
!
!
!
!
!
!!!!!!
!
!
!
!!
!!
!!
!
!
!!!
!
!
!!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!!
!
!
!
!!!
!
!
!
!!
!
!!
!
!
!
!!
!
!
!!!
!
!
!!!!!
!
!!!
!
!
!
!
!
!
!
!!!
!
!
!
!!!
!!
!!
!
!
!!
!
!
!!
!
!
!!
!
!
!!!!!!
!
!
!
!!!!!!
!!
!!!
!
!!!!!
!
!!
!
!
!!!
!
!
!
!!!!!
!
!!!!!!!
!
!!
!!
!!
!
!!
!!
!!!!
!
!
!
!!
!!!
! !
!
!
!
!!!!
!
!!
!
!
!!
!!
!!
!
!
!!
!
!
!
!
!
!!!!
!
!
!
!
!!!
!
!!!
!
!!!!!!!!!!!!!!!!
!
!!!!!
!
!!!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!
!!
!
!
!!
!
!
!
!!!
!
!
!!
!
!
!
!
! !!!
!
!!!!!!
!
!!
!
! !!! ! ! !!! !!!!!!!!!!!
! !
!!!
!
!
!
!
!
!
!
!!!!
!
!
!!!!!!!!!!
!
!!!!
!!
!!!!
!
!
!
!!
!
!
!
!!!!!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!!
!
!
!!
!!!
!
!
!
!
!
!
!!
!!
!
!!!!
!
!!!
!
!
!
!
!!
!!
!
!
!
!!
!
!!!!!!
!
!
!
!
!!!!!
!
!
!!!
!!
!!!!
!
!
!
!
!
!
!
!
!
!
!
!!
!
!!!
!
!!!!!!
!
!!!
!
!!!!!!
!!
!
!
!!
!
!
!!
!
!!!
!
!
!
!
!
!
!!
!
!!!!!!!!!!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!!!
!
!!!!
!
!!
!
!!!
!
!
!
!
!!
!
!
!!!
!
!
!!
!
!
!!
!!
!
!!
!!!
!!
!
!!
!
!!
!
!
!!
!
!
!
!!!!
!
!
!!!
!!
!
!!
!
!!
!
!
!
!
!
!
!
!
!
!!
!!!
!!
!!
!
!
!!
!!!
!
!
!
!
!!
!
!!
!
!
!!
!
!
!!
!
!
!
!!!!
!!
!!!!!!
!
!
!
!
!
!
!!!!!!!
!
!
!!
!
!
!!
!
!
!!
!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!!!!!
!
!!!!
!
!
!
!!
!!
!
!!
!!!!!
!
!!! !!
!
!
!
!
!
!
!
!!
!
!
!
!!
!!
!!
!
!
!
!
!!!!
!
!
!
!!
!
!!!!!!!!!!!
!
!
!
!!
!
!!0
2
4
6
8
!
!
!!!
!
!!!
!
!
!
!!!!
!
!!
!
!
!
!
!
!!!!
!
!!!!!!!
!
!!!
!
!!
!
!
!
!
!
!!
!
!!!!
!
!!!
!
!
!!!!
!
! !!! !! !!!
!
!!!!!!!!! !!
!
!!!!
!!
!
!!!
!
!
!
!
!
!
!!
!
!! !!
!
!! !!
!!
!!!!!!!!
!
!!
!!
!!!
!
!
!
!!
!
!!!
!!!!!!!
!
!
!
!!
!
!
!!
!
!
!!!!!
!
!!!
!
!
!
!!
!
!
!
!!!
!
!!!
!
!
!!!!!!
!
!!!!
!
!!
!
!!
!
!!
!!!!!!!!!!
!
!!!!!
!
!!!!!
!
!!!!
!
!!!!!!!!!!
!
! ! !!!!
!
!
!!!! !
!
!
!
!
!
!
!!!!!
!
!
!
!
!
!!!
!
!!
!
!!!!
!
!!!
!
!
!
!
!!
!!!!!!
!
!!!!!
!
!
!
!
!!!
!!
!!
!
!
!!!
!
!!!
!
!
!
!!
!
!!
!
!!
!
!!
!
!
!
!
!
!
!!!!
!
!
!!!
!
!!!
!
!
!
!
!!!!
!
!
!
!!
!
!
!!
!
!
!
!
!
!
!
!
!
!
!!
!
!
!
!
!
!!
!
!!!
!
!!
!!
!
!
!!
!
!
!
!!
!!!!
!
!
!
!
!
!
!
!
!
!
!
!!!!
!!
!
!
!!!!!!!!
!
!
!!!
!
!
!
!
!!!!!!!
!
!!
!
!
!!!
!
!
!
!!!
!
!!!!!!!
!
!!!!!!!!!
!
!
!
!!
!
!
!
!
!
!!
!
!
!!!
!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!!!
!
!
!
!!!
!
!!!!!!!
!
!
!
!!
!
!!!!
!
!!!
!
!!!
!
!
!!
!
!!!!
!
!
!
!!
!
!!!!!!
!
!!!!!!!!!!!
!!
!
!
!!!!
!
!
!
!!!!!!!!!!!
!
!!!!!!!!!!
!!
!!!!
!
!
!!!!
!
!
!!
!
!!
!
!!!
!
!
!
!
!!
!
!
!
!
!
!
!!!!
!
!!!!
!
!
!
!!!!!!!!!!!!!!!!!
!!
!!!!!
!
!!
!
!!!!!!!!
!
!
!
!
!
!
!
!
!
!
!!
!
!!!!! !!
!
!
!!!!!!!!!
!
!!
!
!
!
!!
!
!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!!!!
!
!
!!!
!
! !!!!! !!
!
!
!!! !!!!!! !!!! !
!
!!! !! !!! !!
!
!
!!!
!!
!
!!!!!!!!!!
!
!
!!!!!!!!
!!
!!
!
!
!
!
!!
!!!!!!
!
!
!
!!!
!
!
!!
!!
!!!!
!
!!
!!
!!!
!
!
!
!
!
!!
!
!!!!!
!
!
!
!
!
!
!
!!!!!
!
!!
!
!
!
!!
!
!
!!!!!!!!!
!
!!!
!
!!!!!!!!!!!!!
!
!
!
!
!
!!!!
!
!!!
!
!!!
!!
!!!
!
!
!! !!
!
!
!!!!!
!
!!!!!
!
!!
!!!
!
!!
!
!
!
!
!
!
!
!
!
!
!
!! !!!!
!
!!
!
!
!
!!!!!!!! !!
!
!! !!0
2
4
6
8
!!!
!
!
!
!!!!!! !!!!!!!!!!!!!!!!
!
!!!!!!
!
!!
!
!!
!
!!!
!
!!
!!
!!
!
!
!
!!!!
!
!!!
!
!!!
!
!!
!
!
!
!
!!
!
!!!! !! !!!! !!!
!
!!!!!
!
!!
!
!!!
!
!
!!!!!!!
!
!
!!
!
!
!!
!
!!!!!! !
!
!!!
!
!!!!!!!
!
!
!!
!!
!!!! ! !
!
!!
!
!!!
!
!
!!!
!
!!
!
!!!!!!!!!!!!!!!!
!
!
!
! !!! !
!
!
!
!
!
!!! !
!
!!
!
!
! !!! !!!!
!
!! !!!!
!
!
!
!
!!!
!
!!!!!
!!
!
!
!!!!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!!
!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!!
!
!
!
!
!!!
!
!!
!
!
!
!!
!!
!
!!
!
!
!
!
!!!
!
!
!!!!
!
!!!
!
!!!!!!!
!!!
!
!
!
!
!
!!!
!
!
!!!
!
!
!
!
!
!!
!
!
!!!!!!
!
!
!
!
!!!!!!!!!!
!!
!!
!
!!!!
!
!
!
!!!
!
!! !!!!
!
!
!
!
!!!!
!
!!
!
!
!
!!!!!
!
!!
!
!
!
!
!
!!!!!!!!
!!
!
!
!!!!!!!
!
!!
!
!
!
!
!
!
!
!
!!
!
!
!
!!
!
!
!
!!!!
!
!!!
!!
!!!
!
!
!!
!
! ! !!!! !!!!!!!!!!!!
!
!!!!!!!!!!!!!
!
!!!
!
!!
!!!!!! !!!!! !!!!!
!
!!!!!!!
!
!!
!
!!!
!
!!
!
!!
!!
! !!!!!
!
!!!
!
!!!
!
!
!
!
!
!
!
!!
!
!! !!
!
!!!
!
!! !!!!!!!!
!!
!
!
!
!
!!!
!
!!!!!!!!!
!
!
!
!
!
!!
!
!
!
!
!! !!
!
!! ! !! !!!
!
!!! !
!!
!!
!
!
!
!!
!!
!
!
!
!
!
!!
!
!!
!
!
!!!
!
!
!
!!!!!!!!!
!
!
!!
!!
!!
!!!!
!
!!
!
!
!
!!
!
!!
!
!!!!!!!!!!!!!!!!!
!
!!!
!
!
!
!
!
!!!!
!!
!
!
!
!
!
!
!!
!
!
!
!!! ! !
!
!
!
!!!!!
!
!
!
!!
!!
!
!!!!!!
!
!!!!!!!!!!!!!!!!!!!!!!!!
!
!
!
!
!
!
!!!
!
!!
!!
!!
!
!!
!
!
!
!!!!!
!
!
!
!
!!
!
!
!!
!
!
!!
!!!
!
!
!
!
!
!
!!
!
!
!
!!
!!
!!
!
!
!
!
!
!
!!!!!!
!
!!!!
!
!!!!
!!
!
!!
!
!
!
!!!
!
!
!
!
!!!!
!
!!
!
!
!!
!
!!!
!!
!
!
!
!!!!!!
!
!!!!
!
!!! !! !
!
!!!
!
!
!
!!!
!! ! !!!!!!
!
0
2
4
6
8
!!!!!!!
!
!!!
!
!!
!!!
!
!
!!!!!
!
!!
!
!
!!!!!!
!
!
!!!
!!
!
!!!!
!
!!
!
!!!!
!!!!!
!!
!!!!
!
!
!!!!!!!
!!!!
!!!
!!!!
!
!!!
!
!!!
!
!
!
!
!
!
!!
!
!!!
!
!!
!!
!!
!
!!
!
!
!
!
!
!
!!!!
!!
!!!
!
!!!
!!
!
!
!
!
!!
!
!!!
!
!
!
!
!!!
!
!
!
!
!
!
!
!
!
!
!!!!
!
!
!
!
!
!
!
!
!
!!
!
!!
!
!
!
!
!!!
!
!
!
!!
!
!!!!
!
!
!!
!
!
!
!!
!
!
!
!!
!!
!
!
!
!
!
!!
!
!
!
!
!
!
!
!
!
!!!
!
!
!
!!
!
!!
!!
!
!!!
!!
!!
!!!
!!!
!!
!!
!!
!
!
!!
!
!!!!!
!!!
!!!
!!!!
!
!!
!!
!!
!
!
!
!!!!
!!
!!
!!!
!
!
!!
!
!
!
!
!
!!!
!!!!
!
!
!
!
!!!
!
!
!
!
!!
!
!
!!
!
!
!
!
! !
!
!!
!3!2!1
0123
!
!
!
!
!!!!!! !!!!
!
! !!!!!!!!!!!!!!!! ! !
!
!! !!! ! !!!!!!!! !!!!!!
!
!!!!!!!!!!
!
!
!
!
!
!
!
!!!!!!!!!
!
!
!
!!
!
!!!!!!
!
!!!
!
!!!!
!
!!!
!
!!!!!!!
!
!!!! !!
!
!!
!!!!
!
!!!
!!!!!!!
!
!
!!!
!
!
!!!
!
!!
!!!
!
!!
!
!
!
!
!!
!
!!
!
!
!!
!!
!!!!!!!
!!!
!!!!!!!!
!
!
!!
!!
!
!!!!!!!
!
!!!!!!! !!!!
!
!!!!!!!!!!!!!!!!!
!
!!!
!
!!!!!!!
!!
!!!!!!!
!
!!!!!!!!!!!!
!
!!
!!
!
!
!!!!!!
!!
!!!
!
!
!
!!
!
!!!
!!
!
!
! !!!!!
!
!!!
!
!!
!
!!
!
!!
!
!
!!!!!
!!!
!
!!!!!!!!!!!
!
!!!!!!! !!
!
!!!!!! !!!!
!
!!
!
!!!!!!!
!!
!!!!!
!
!!
!
!!!!!!!!!!!!!!
!
!!!!!!!!
!
!
!!
!!!!!!!!
!
!
!
!!!!!! !!
!
! !! !
!!!
!!!
!!
! !!
!!!!
!!!!!!
!
!!
!!
!
!
!
!
!!!
!
!!!!
!!
!!!!!
!!
!
!!!!!
!
!
!
!
!
!
!!!!!!!!
!
!
!
!!!
!
!!!!!!!!!!!
!
!
!
!!!!!!
!
!!!
!!!
!!
!
!
!
!!!
!
!
!
!!
!
!
!
!
!
!
!
!!
!
!!!!!!!
!
!
!!
!
!
!
!
!!
!!!
!!
!!!!
!
!
!
!!!
!
!
!!!!!!
!
!
!!
!
!!!
!
!
!!!!!!
!!!!
!
!
!
!!
!
!!
!
!
!!!!
!
!!
!
!!!!!!
!
!!!
!
!!!!!!
!!
!!!!
!!
!!!!!!
!!
!!
!
!!!!!
!!
!!!
!
!!
!
!
!
!!!!
!
!!
!!!!!!!!!!!!!!!!
!!
!
!
!
!
!!
!
!
!
!
!
!!!!!
!!!
!
!!
!!
!!!!
!!!
!!!!
!
!!!
!
!!!
0
2
4
6
8
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!
!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!
!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!
!4!3!2!1
012
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
0.3196
0.32
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!
!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!
!!!!!!!
!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!
!!
!
!
!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!
!
!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!
!
!!!
!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!
!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!
!
!!
!
!!!!!!!!!!
!
!!!!!!!!!!!!!!
!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!
!
!
!
!
!
!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!
!!!!!!
!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!
!
!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!
!!!!!!!!
!!!!!!!!!!!!!!!!!
!!
!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!
!
!
!!
!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!
!
!!
!!!!!!
!!!!!!!!!!!!!!
!
!!!
!!
!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!
!
!
!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!
!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!
!
!
!!!!!!!!!!!!!!!!!!!!!!!
!
!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
00.20.40.60.8
11.2
mcl.9, chr: 5
Acknowledgements
Statistics
Jim BullardSandrine DudoitElizabeth PurdomMargaret TaubSteffen DurinckTerry Speed
RNA assembly
Cole Trapnell
S. Cerevisiae
Gavin SherlockAlbert Lee
D. Melanogaster
Brenton GravelyMike DuffLi YangSteven BrennerAngela Brooks
top related