A high-resolution map of transcription in the
Lior David*†, Wolfgang Huber†‡, Marina Granovskaia§, Joern Toedling‡, Curtis J. Palm*, Lee Bofkin‡, Ted Jones*,
Ronald W. Davis*¶, and Lars M. Steinmetz*§¶
*Stanford Genome Technology Center and Department of Biochemistry, Stanford University, Palo Alto, CA 94304;‡European Bioinformatics Institute,
European Molecular Biology Laboratory, Cambridge CB10 1SD, United Kingdom; and§European Molecular Biology Laboratory, 69117 Heidelberg, Germany
Contributed by Ronald W. Davis, February 10, 2006
There is abundant transcription from eukaryotic genomes unac-
counted for by protein coding genes. A high-resolution genome-
wide survey of transcription in a well annotated genome will help
relate transcriptional complexity to function. By quantifying RNA
expression on both strands of the complete genome of Saccharo-
myces cerevisiae using a high-density oligonucleotide tiling array,
this study identifies the boundary, structure, and level of coding
and noncoding transcripts. A total of 85% of the genome is
operon-like transcripts, transcripts from neighboring genes not
separated by intergenic regions, and genes with complex tran-
scriptional architecture where different parts of the same gene are
expressed at different levels. We mapped the positions of 3? and
5? UTRs of coding genes and identified hundreds of RNA transcripts
average, have lower sequence conservation and lower rates of
deletion phenotype than protein coding genes. Many other tran-
pairs global correlations were discovered: UTR lengths correlated
with gene function, localization, and requirements for regulation;
antisense transcripts overlapped 3’ UTRs more than 5’ UTRs; UTRs
with overlapping antisense tended to be longer; and the presence
of antisense associated with gene function. These findings may
suggest a regulatory role of antisense transcription in S. cerevisiae.
Moreover, the data show that even this well studied genome has
transcriptional complexity far beyond current annotation.
tiling array ? transcriptone survey ? gene architecture ? segmentation ?
also the main controllers of cellular processes. Recent evidence
challenges this assumption, suggesting a wide-spread involvement
of noncoding RNA in regulation, including through the activity of
isolated noncoding RNAs such as microRNA that control tran-
script levels or their translation (4).
High-resolution transcriptome analysis in higher eukaryotes
using tiling arrays has improved ORF annotations and exon-
intron predictions and discovered many new transcripts of
currently unknown function (5–7). However, these studies have
encountered challenges, due to noise, limited resolution, lack of
strand-specific signal, and drawbacks in the analysis methods (8).
Sequencing of cloned cDNAs has also revealed a high level of
transcriptional complexity, including the presence of many new
transcripts, alternative promoter usage, splicing, and polyade-
nylation, as well as the presence of many sense–antisense
transcript pairs (3, 9). However, because of the cost and labor of
large-scale sequencing, this approach has been limited. There-
fore, there is a need to develop high-throughput, precise, and
high-resolution technology to map the full transcriptional activ-
ity. Yeast is a simple and relatively small eukaryotic genome that
provides opportunities to rapidly characterize novel findings.
We developed an oligonucleotide array for Saccharomyces cer-
enables a 4-nt resolution for hybridization of double stranded
targets and an 8-nt resolution for strand-specific targets. We
profiled transcription during exponential growth in rich media, the
map of transcription.
Results and Discussion
Microarray Experiments and Analysis. We hybridized first-strand
cDNA synthesized using random primers from polyadenylated
[poly(A)] and total RNA. To calibrate the sequence-specific
probe effect (10–12), we background-corrected and adjusted
(13) the signal of each probe by sequence-specific parameters,
estimated from a calibration set of genomic DNA hybridizations
(Fig. 5, which is published as supporting information on the
PNAS web site). This method allowed us to quantitatively
compare the signal from probe to probe on the array.
The Transcriptome. To address the question of how much of the
genome is transcribed, we analyzed the coding regions of 5,654
ORFs that were annotated as verified or uncharacterized genes
in the Saccharomyces Genome Database (SGD, www.yeastgeno-
me.org) and represented by unique probes on the array. Signif-
icant expression above background was detected for 5,104 ORFs
(90%) (Binomial test, false discovery rate ? 0.001; Fig. 6, which
is published as supporting information on the PNAS web site).
As expected, genes that were not detected have functions not
required in this condition such as meiosis, sporulation, mating,
sugar transport, and vitamin metabolism [hypergeometric test
for gene ontology (GO) annotation enrichment, unadjusted P ?
3 ? 10?9]. In addition, analyzing 11,412,997 bp of unique
genomic sequence, we detected expression above background on
either strand for 85%. Comparing this to existing annotation,
which covers ?75% of the genome, shows that 16% of the
transcribed base pairs had not been annotated before.
To obtain an unbiased map of the position, abundance, and
architecture of transcripts, the hybridization signals were examined
along their chromosomal position for each strand (Fig. 1). The
profiles were partitioned into segments of constant hybridization
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviation: GO, gene ontology.
Data deposition: The array data have been deposited in ArrayExpress database (accession
†L.D. and W.H. contributed equally to this work.
¶To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or larsms@
© 2006 by The National Academy of Sciences of the USA
April 4, 2006 ?
vol. 103 ?
aries. We used a change point detection algorithm that determines
the global maximum of the log-likelihood of a piece-wise constant
model by dynamic programming (14, 15). Compared to running-
window approaches, it finds more accurate estimates of change
point locations and depends on fewer user-defined parameters.
Segments were determined separately for poly(A) and total RNA
(Tables 3 and 4, respectively, which are published as supporting
information on the PNAS web site). Segments from poly(A) and
total RNA were remarkably concordant, and many noncoding
is published as supporting information on the PNAS web site).
Overall, the poly(A) RNA hybridization data were cleaner and
therefore were the focus of our analysis.
The automated segmentation algorithm provides an unbiased
global analysis, but the data complexity invites additional man-
ual curation. Profiles for all genomic regions are provided in a
database that is searchable by gene symbol or chromosomal
coordinate (www.ebi.ac.uk?huber-srv?queryGene). We encour-
age readers to explore the database along with the examples
hybridization data accurately separates exons from spliced introns,
with more than one perfect match in the genome are colored gray. Annotated ORFs are shown as blue boxes, dubious ORFs are shown as light blue boxes, and
transcription factor binding sites are shown as gray bars. Vertical lines are segment boundaries. The background threshold (y ? 0) is shown as a horizontal line.
Visualization of yeast tiling array intensities along 100 kb of chromosome 1, corresponding to ?1% of the genome. The plot shows the normalized
Complex transcript architecture of MET7. (d) Overlapping transcripts of two ORFs. (e) Adjacent transcripts of SER3 and the noncoding SRG1. (f) Nonannotated
isolated transcript. (g) Transcript antisense to SPO22. CDS refers to coding sequence; uORF, upstream ORF; ncRNA, noncoding RNA; TF, transcription factor. Plot
layout as in Fig. 1.
Examples of transcriptional architecture. (a) Detection of spliced transcripts. (b) Long 5? UTR of GCN4 including its cotranscribed upstream ORFs. (c)
David et al.
April 4, 2006 ?
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shown for RPS16A (top strand) and RPL13B (bottom strand) (Fig.
2a). The segmentation-mapped UTRs of coding transcripts (e.g.
GCN4, Fig. 2b) identified complex transcriptional architectures,
such as uneven transcript levels for different regions of a single
ORF (MET7, Fig. 2c), determined transcripts spanning multiple
ORFs (YCK2, GIM3, Fig. 2d), and identified adjacent transcripts
for neighboring genes, uninterrupted by untranscribed intergenic
regions (SRG1, SER3, Fig. 2e). Moreover, we observed transcripts
in regions of the genome lacking prior annotation (Fig. 2f), as well
as transcripts opposite annotated features (Fig. 2g). For each of the
UTR Boundaries. To map UTRs, we compared ORF boundaries
with segment boundaries. We automatically determined UTR
lengths for verified or uncharacterized nuclear-encoded genes
whose annotated coding sequence was fully contained within a
single segment. A total of 2,223 segments passed a confidence
filter that required a sharp decrease in signal on both sides of the
segment. UTR coordinates are given in Tables 3 and 4. We
proceeded with analysis of the 2,044 poly(A)-determined UTRs
because the poly(A) hybridization data were cleaner and yielded
most of the UTR determinations (Fig. 7, which is published as
supporting information on the PNAS web site). For many
can be mapped by closer inspection.
We found that 3? UTRs were significantly longer than the 5?
UTRs, with a median of 91 vs. 68 nt (Fig. 3a). Longer 3? UTRs
are consistent with them containing posttranscriptional regula-
tory regions that influence mRNA stability, localization, and
translation (16), and with findings from other species (17). The
mean sum of 3? and 5? UTR lengths was 211 nt and similar to a
mean of 256 nt found by a gel-mobility assay (18). We computed
662 3? UTRs from ESTs (19) and compared them to 435 ORFs
that had UTRs in our data set. The Pearson correlation coef-
ficient between the UTR length estimates was 0.63. A contri-
bution to the differences is that in the EST data the longest
transcript was chosen, whereas the array measures the average
transcript abundance at each probe position.
We compared UTR lengths with transcript levels and coding
sequence (ORF) lengths. Although transcript level was generally
nor ORF lengths were significantly associated with UTR lengths.
We also compared length distributions of UTRs for different
functional and localization categories (GO annotations) and de-
tected significant correlations (Fig. 3b). The longest 3? UTRs were
found for transcripts of proteins that are targeted to the mitochon-
drial electron transport chain, the plasma membrane, and the cell
wall. These longer 3? UTRs may contain mRNA localization
signals, as has been well demonstrated for mitochondrial targeted
proteins (20, 21). Genes involved in phosphorylation, transporter
activity, ion transport, and specific stages of the mitotic cell cycle
had both ends longer. Genes involved in RNA processing, rRNA
fore, genes with longer UTRs seem to fall into categories that
categories with a reduced need for posttranscriptional regulation,
such as housekeeping genes.
Complex Transcriptional Architectures. Many expressed segments
flanked other expressed segments with different signal levels,
thus making up complex transcriptional architectures. In many
cases, different parts of the same gene are expressed at different
levels: 921 ORFs from the poly(A) RNA sample were divided
into at least two expressed segments, one covering ?50% of the
feature and others ?50%. Such complex architectures could be
due to alternative transcription initiation, termination, or alter-
human genes (22). In yeast, it has been suggested that up to 20%
of mRNAs have alternative 3? ends (23). Complex hybridization
patterns on the array could also be caused by RNA decay or
variation introduced by reverse transcription, because the array
captures the sum of cDNA molecules present at the time of
hybridization. The explanation of our observations by such
mechanisms will require a case-by-case analysis.
architecture matches previous results describing alternative 3? ends
in response to carbon source regulation (24). For GCN4, lower
hybridization signal was observed at the 3? end (Fig. 2b). GCN4 is
not translated during nutrient-rich growth because of the transla-
tion of the upstream ORFs encoded in the same transcript (25). 3?
3? end signal. In support, this decrease was not seen in an oligo(dT)
reverse-transcribed sample, where no priming would occur on
degraded poly(A) transcripts. At the 5? end, the segment boundary
matches the previously determined position to within nine bases.
length. Analyses were based on 2,044 genes from poly(A) samples. (a) Scat-
terplot and histogram of 3? vs. 5? UTR lengths. (b) Association between UTR
length, cellular localization, and biological process. Length distributions be-
tween genes inside and outside of GO categories were compared, and se-
lected significant categories are shown (orange, cellular component; green,
biological process; blue, molecular function). For each category, a horizontal
line shows the 5? and 3? median UTR lengths measured in nucleotides (x axis).
The median over all genes is shown by a vertical dashed line. Significant
medians are indicated by asterisks, red longer, blue shorter (two-sided Wil-
coxon test, P ? 0.002).
Length of UTRs and functional categories with exceptional UTR
www.pnas.org?cgi?doi?10.1073?pnas.0601091103 David et al.
For MET7, the annotated gene was segmented into three regions
(Fig. 2c), suggesting a misannotation of the translation start site. A
later transcription start site is supported by the multiple sequence
alignment of yeast species in SGD, which shows that conservation
of MET7 starts at a later methionine (M55), whose position agrees
difference between the central and the 3? segment was not seen in
a poly(A) sample that was reverse transcribed by using oligo(dT)
PNAS web site), consistent with early transcript termination or
RNA decay. Altogether, we tested 27 regions from 10 genes by
PCR results matched the architectures in the array data (Fig. 8).
Neighboring Transcription. Additional unusual architecture was
found for adjacent ORFs not separated by an unexpressed region.
Such architectures can result from more than one ORF being
or from distinct transcripts not separated by untranscribed inter-
genic regions. We found the ORFs of GIM3 and YCK2 within one
segment resembling a bicistronic transcript (Fig. 2d). The Phast-
Cons multiple alignment (26) of the intergenic region with other
yeast species shows high sequence conservation, but includes
frame-shifting gaps, which suggests that the two ORFs are not
translated as one. By reverse-transcription PCR across the gap
between the ORFs, a product was obtained supporting either
reported for few eukaryotic species and mostly for Caenorhabditis
elegans (27). A bicistronic transcript had been reported previously
in yeast for YMR181C and RGM1 (28), and we observed different
transcript levels for the ORFs, but no separation by an untran-
Fig. 2e shows two other adjacent transcripts, SRG1 and SER3,
expressed at different levels and not separated by an intergenic
region. It had been proposed that SRG1, an upstream noncoding
RNA, represses the expression of SER3 in rich media, by
reducing the binding of SER3 transcription factors (29). In
contrast, we find that SER3 is expressed significantly above
background, suggesting that even though SRG1 is expressed at
transcribed. There are many cases of adjacent genes not sepa-
rated by unexpressed, intergenic regions in our data set, and this
suggests that transcription over active promoters of adjacent
genes is common in yeast. Some further examples are QCR6,
PHO8, RIB3, HCH1, UBI4, SEC53, RPS26A, and ADE13.
Unannotated Transcripts. Many segments with signal above back-
ground did not overlap existing annotation. They fall into two
classes: nonannotated isolated segments if there was no prior
annotation on either strand (Fig. 2f), and nonannotated anti-
sense segments if there was an annotation on the opposite strand
(Fig. 2g). Many are not independent transcription units: some
represent UTRs of genes with complex transcriptional architec-
ture; others are part of unannotated transcripts that are divided
into multiple segments. The identification of antisense tran-
scripts requires caution because reverse transcription can gen-
erate double stranded cDNA from secondary mispriming (30,
31). Considering these concerns, we applied a filter requiring
segments to be at least 48 bp long, be flanked by segments with
reduced hybridization signal on both sides, and have higher
expression signal than seen on the opposite strand for at least
part of their length. In these filtered categories, we obtained 427
nonannotated segments from poly(A) and 357 from total RNA
hybridizations. These segments divide approximately equally
into the isolated and antisense categories (Fig. 4a). Antisense
segments and segments overlapping annotation (?50%) had
similar length distributions and tended to be longer than those
of the isolated categories (Fig. 4b). Isolated segments showed
similar levels of expression as annotated segments, whereas
antisense segments had lower expression levels (Fig. 4c).
Nonannotated Isolated Transcripts. We verified the array identifica-
tion of 126 nonannotated isolated transcripts by RT-PCR. All were
expressed in both total and poly(A) RNA reverse transcribed by
using random and oligo(dT) primers, respectively. For 10 of them,
a quantitative real-time PCR analysis showed their levels to be
similar to expressed ORFs in both sample types.
The 1.7-kb transcript between ORC2 and TRM7 (Fig. 2f) is an
example of a nonannotated isolated segment that is highly
conserved across other yeast species in the PhastCons multiple
alignment (26). Nevertheless, assessment of 125 nonannotated
isolated segments showed that only 48 had a multiple alignment
?50 nt across four yeast species (32). In addition, median total
tree lengths of the phylogenies were 0.59 for verified genes, 1.08
for undetected unannotated segments, and 1.50 for nonanno-
tated isolated segments (Table 6, which is published as support-
ing information on the PNAS web site). To assess protein-coding
potential, we tested for dissimilarity in evolutionary rates among
first, second, and third codon positions in all reading frames.
There was no protein coding signature for the 48 nonannotated
segments (median likelihood-ratio statistic of 1.06, compared to
1.14 for undetected unannotated segments and 162.0 for verified
genes). Conservation of DNA sequence or protein coding ability
is nevertheless neither a necessary nor a sufficient attribute of
We generated knockouts for 47 nonannotated isolated segments
levels. (a) Number and percentage of the expressed segments detected from
the poly(A) RNA and total RNA hybridizations. Categories ‘‘?? 50%’’ and
‘‘?50%’’ consist of segments that overlap more, or less, than half of an
annotated feature, respectively. The ‘‘nonannotated isolated’’ category con-
sists of segments that have no overlap with annotated features on either
of the high confidence segments that passed our filter, and the ‘‘unassigned’’
categories consist of the remaining segments. Length (b) and transcript level
(c) distributions for segments from the above categories are given.
Categories of expressed segments, their length, and their expression
David et al.
April 4, 2006 ?
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and tested for growth defects in rich media conditions (Table 7,
which is published as supporting information on the PNAS web
site). A growth defect was identified for two knockouts: one on
chromosome 6, positions 54813–55221, the other on chromosome
7, positions 622039–622295. On chromosome 6, the deleted seg-
ment contained annotated transcription factor binding sites up-
stream to ACT1, an essential gene, which likely accounts for the
observed inviability. On chromosome 7, the deletion does not
overlap any annotation, and strains with deletions of the neighbor-
segment does not appear to be evolutionarily conserved or to
the 47 knockouts is much lower than the ?40% found for knock-
outs of protein-coding genes (33).
Nonannotated Antisense Transcription. We identified antisense
transcripts opposite to 1,555 genes, of which 402 were in the
filtered set from both poly(A) and total RNA samples (Tables 3
and 4). The antisense transcripts are not caused by read-through
from ORFs on the opposite strand, but appear as independent
transcription units. For example, antisense transcription was
found opposite SPO22, a meiosis-specific protein induced early
in meiosis (Fig. 2g). Upstream of this antisense transcript, there
replication and chromosome cycle and is important for growth
in rich media, suggesting that the antisense expression may be
negatively correlated with the expression of SPO22.
Many genes with antisense transcripts had products that localize
to the cell cortex and cell wall, and that function in the meiotic cell
cycle and in transcriptional regulation (Table 1). Some of these
categories included genes not active during growth in rich media,
like meiosis. Others included genes that are active during growth in
rich media, but which may need posttranscriptional regulation.
Further correlations were found between UTRs and their opposite
antisense segments: More antisense transcripts overlapped the 3?
transcripts were longer than UTRs that did not (Table 2).
The generation and significance of the many nonannotated
transcripts is unclear. Regulation of gene expression by antisense
transcripts was reported in prokaryotes (34) and higher eu-
karyotes (35). Sense?antisense transcript pairs were suggested to
be frequent in mammalian genomes and to provide regulatory
function (3). In S. cerevisiae, major components of the RNA
interference machinery have not been identified (36); however,
in other species, alternative mechanisms for regulation by non-
coding RNAs exist (2, 37). In Drosophila, it has been shown that
microRNA predominantly target 3? UTRs (38) and these UTRs
also tend to be longer than UTRs of genes not targeted (39). We
observed similar correlations for antisense transcripts in S.
cerevisiae, which together with their association to particular
functional categories may suggest a possible regulatory role.
There are experiments supporting this hypothesis: artificial
antisense transcripts in S. cerevisiae had effects on expression of
several genes (40–42), and overexpression of random genomic
fragments antisense to ORFs has led in several cases to growth
inhibition (43). In our data set, naturally occurring antisense
transcripts were found for ?20 of these cases.
Most ncRNAs previously reported as novel have since been
annotated in SGD, and hence do not overlap with our expressed,
nonannotated segments (44, 45). We compared our data to
expression (SAGE) (46) and ESTs (19). Thirteen percent of the
nonannotated isolated and 42% of the nonannotated antisense
transcripts were represented by SAGE tags. For the EST data,
these numbers were 1% and 6%, respectively. Analysis of SAGE
tags on microarrays described a number of novel transcripts in
a mutant strain defective in the RNA degradation pathway (47);
however, the eight primary examples were not found expressed
in our study of wild-type yeast.
This study reveals considerable transcriptional activity in yeast
that is currently not systematically annotated. Our transcription
map will be useful for annotating the genome. Furthermore, the
position of transcription initiation and termination sites will help in
defining the promoters and transcriptional regulators of genes.
Although our results suggest that not many new, long protein-
coding regions will be discovered in yeast, the extensive noncoding
transcription detected in regions with no prior anotation and
antisense to annotated transcripts invites further investigation.
Therefore, even for a genome that has been studied intensively
since it was sequenced 10 years ago (48), a glimpse into the
complexity of its transcriptional architecture makes this genome
appear like novel territory.
Materials and Methods
Array Design and Sample Hybridization. The array was designed in
collaboration with Affymetrix (Santa Clara, CA) (PN 520055).
An S288c background strain S96 (MATa gal2 lys5) was grown in
rich yeast-extract?peptone?dextrose media to mid-exponential
Table 1. Selected GO categories found overrepresented among the 355 genes opposite
filtered nonannotated antisense segments
M phase of meiotic cell cycle
Transcriptional activator activity
Transcriptional repressor activity
Monosaccharide transporter activity
2 ? 10?6
9 ? 10?7
5 ? 10?6
1 ? 10?4
3 ? 10?4
that were opposite an antisense segment; Nexp, number of genes expected if genes opposite antisense segments
are randomly distributed over GO categories; P, hypergeometric test P value.
Table 2. Association of UTR lengths with presence of antisense
transcript, and the 3??5? bias in position of antisense transcripts.
Number of 3? overlaps
Number of 5? overlaps
Median length of 3? UTR
(no. of genes)
Median length of 5? UTR
(no. of genes)
P ? 0.05
P ? 0.08
P ? 0.00001
P ? 0.003
Overlap was measured with respect to the start and stop codons. Signifi-
cance was calculated by comparing the length distributions of UTRs with
antisense to controls where UTRs had no antisense partner by using the
two-sided Wilcoxon test. NA, not applicable.
www.pnas.org?cgi?doi?10.1073?pnas.0601091103 David et al.
RNA was enriched by two rounds of the Oligotex mRNA kit Download full-text
(Qiagen). First-strand cDNA was synthesized by using random
primers. Three replicate hybridizations (biological) of poly(A),
two of total RNA, and three of genomic DNA were performed.
Probe Annotation. Probe sequences were aligned to the genome
sequence of S. cerevisiae strain S288c (SGD of August 7, 2005).
Perfect match probes were further analyzed.
Normalization. RNA hybridization intensities were adjusted by
where Xijis the RNA intensity of the ith probe on the jth array,
Ai is the geometric mean of the intensities from the DNA
hybridizations, Bj(A) is a continuous function that parameterizes
the estimated background of probes with gain A, and Nijis the
adjusted intensity. Probes were grouped into 20 strata defined by
the 5%, 10%, 15%, . . . , 100% quantiles of Ai. Within each
stratum, and for each array j, the midpoint of the shorth of the
intensities of the probes for which no genomic feature was
annotated on either strand was calculated. Linear interpolation
yielded the function Bj. Dead probes (the 5% of probes with
lowest signal in the DNA hybridization) were discarded. The
values Nij were background-adjusted and transformed to log2
scale by using VSN (13). Fig. 5 demonstrates the successive
improvements in the signal-to-noise ratio during normalization.
Segmentation. Segments of approximately constant hybridization
signal were defined by using a dynamic programming algorithm
that, for each chromosome strand separately, minimizes the cost
G?tl, . . . , ts? ??
?yij? y ?sj?2,
where yijis the VSN-normalized signal of the ith probe on the jth
replicate array, y ?sjis the arithmetic mean of the signal values of
array j in segment s, S is the number of segments, and t1, . . . , tS
are the segment boundaries (15). For each chromosome, S was
chosen such that the average segment length was 1,500 nt. S, the
only parameter of the segmentation algorithm, controls the
sensitivity–specificity tradeoff and was chosen to yield high
All analyses were performed with custom-written software in
the language and statistics environment R (49) and BIOCONDUC-
TOR (14). For additional details on analyses and experimental
procedure, see Supporting Text, which is published as supporting
information on the PNAS web site.
We thank Mike Mittmann for the array design, Iain Russell and
Victor Sementchenko for experimental advice, Raquel Kuehn and
Michelle Nugyen for technical assistance, Roy Parker, Rafael Irizarry,
Elisa Izaurralde, Steve Cohen, and Nick Goldman for helpful comments
on the manuscript, and contributors to the BIOCONDUCTOR (www.
This work was funded by the National Institutes of Health (R.W.D and
L.M.S.) and the Deutsche Forschungsgemeinschaft (L.M.S.).
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