Partitioning Transcript Variation in Drosophila:
Abundance, Isoforms, and Alleles
Yajie Yang,*,†Rita M. Graze,*,†Brandon M. Walts,* Cecilia M. Lopez,*,†Henry V. Baker,*,†
Marta L. Wayne,*,‡Sergey V. Nuzhdin,** and Lauren M. McIntyre*,†,§,1
*Genetics Institute, University of Florida, Gainesville, FL 32610-3610,†Department of Molecular Genetics and
Microbiology, University of Florida, Gainesville, FL 32610-0266,‡Department of Zoology, University of Florida,
Gainesville, FL, 32611-8525,§Department of Statistics, University of Florida, Gainesville, FL 32611-8545, and
**Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089-2910
ABSTRACT Multilevel analysis of transcription is facilitated by a new array design that includes modules for
assessment of differential expression, isoform usage, and allelic imbalance in Drosophila. The ?2.5 million
feature chip incorporates a large number of controls, and it contains 18,769 39 expression probe sets and
61,919 exon probe sets with probe sequences from Drosophila melanogaster and 60,118 SNP probe sets
focused on Drosophila simulans. An experiment in D. simulans identified genes differentially expressed
between males and females (34% in the 39 expression module; 32% in the exon module). These proportions
are consistent with previous reports, and there was good agreement (k ¼ 0.63) between the modules.
Alternative isoform usage between the sexes was identified for 164 genes. The SNP module was verified
with resequencing data. Concordance between resequencing and the chip design was greater than 99%.
The design also proved apt in separating alleles based upon hybridization intensity. Concordance between
the highest hybridization signals and the expected alleles in the genotype was greater than 96%. Intrigu-
ingly, allelic imbalance was detected for 37% of 6579 probe sets examined that contained heterozygous
SNP loci. The large number of probes and multiple probe sets per gene in the 39 expression and exon
modules allows the array to be used in D. melanogaster and in closely related species. The SNP module can
be used for allele specific expression and genotyping of D. simulans.
Gene expression analysis has proceeded from a primary focus on
overall transcript level (Schena et al. 1995; Ross et al. 2000; Rifkin
et al. 2003) to more sophisticated analyses, including those that ex-
amine expression of different isoforms (Johnson et al. 2003; Kwan
et al. 2008) or individual alleles (Lo et al. 2003; Zhang et al. 2009).
Commercial platforms exist for measuring 39 expression or exon
expression; however, there is not a single cost-effective platform for
measuring expression at multiple levels. This article presents an array
with three modules: 39 expression, exon, and SNP probes for Drosophila.
In diploid organisms, expression from two, potentially different,
copies of each gene contribute to transcript level and subsequent
protein production. Unequal expression of these alleles is termed
allelic imbalance (AI). AI is observed in model organisms and humans
(e.g., Lo et al. 2003; Guo et al. 2008; Graze et al. 2009; Zhang and
Borevitz 2009). AI is a factor in predisposition to complex diseases
(Meyer et al. 2008; de la Chapelle 2009) and contributes to phenotypic
variation in human populations (Johnson et al. 2005; Pickrell et al.
2010). For example, AI is associated with the risk of developing breast
cancer (Meyer et al. 2008) and colorectal cancer (de la Chapelle 2009).
AI has a genetic (as well as epigenetic) basis (e.g., Pastinen et al.
2004; Serre et al. 2008; Wang et al. 2008; Verlaan et al. 2009). Exciting
new developments in the study of complex diseases revealed regula-
tory polymorphisms contributing to the evolution of gene regulation
(e.g., Emerson et al. 2010). Whole-genome associations of gene ex-
pression and phenotype identify the genetic basis of disease and other
Copyright © 2011 Yang et al.
Manuscript received June 15, 2011; accepted for publication September 11, 2011
This is an open-access article distributed under the terms of the Creative
Commons Attribution Unported License (http://creativecommons.org/licenses/
by/3.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Supporting information is available online at http://www.g3journal.org/lookup/
Arrays have been submitted to the GEO database at NCBI as the custom
platform GPL11273 and series GSE31750. Sequencing data have been submitted
to the SRA database at NCBI as SRP005952.
1Corresponding author: 2033 Mowry Road, Cancer/Genetics Research Complex,
University of Florida, Gainesville, FL 32610-3610. E-mail: firstname.lastname@example.org
Volume 1|November 2011|
important phenotypic variation (Stranger et al. 2007; Nica and Der-
mitzakis 2008; Nica et al. 2010). AI identifies causal cis regulatory
variants (Wittkopp et al. 2004). Allele-specific association studies ad-
vance these analyses and increase scientific knowledge of the regula-
tory process (Rockman and Kruglyak 2006; Serre et al. 2008;
Stamatoyannopoulos 2004). Analysis of AI is an important next step
in identifying the genetic basis of expression differences.
AI has been assayed with pyrosequencing (Ahmadian et al. 2000;
Wittkopp et al. 2004), targeted SNP typing arrays (e.g., Serre et al.
2008), high-density array designs (e.g., Zhang and Borevitz 2009),
RNA-Seq–based methods (Zhang et al. 2009 ; McManus et al. 2010;
Pickrell et al. 2010), and smaller-scale methods, such as allele-specific
qPCR (Szabó and Mann 1995).
This article presents a custom array for measuring 39 expression,
exon expression (and thus alternative splicing), and AI. The array has
been designed for Drosophila on an Affymetrix platform (UFL Cus-
tom Dros_snpa520726F Array Format: 49-7875; available for pur-
chase from Affymetrix). The use of a single platform is cost
effective, and statistical analysis is simplified by the single hybridiza-
tion. We designed 60,118 D. simulans SNP probe sets from previously
reported SNP variants (Benson et al. 2005; Begun et al. 2007). In total,
these probe sets allow AI to be assessed for 11,929 genes [79% of
15,107 genes in FlyBase R5.11 (August 2008)], with the majority of
genes represented by multiple SNP probe sets. The SNP module is
complemented by two additional modules: one that measures 39 ex-
pression and another that analyzes exon-level expression concurrently
with allele specific expression (ASE). Experiments show an amount of
sex bias (34% of 18,769 probe sets), alternative exon usage (164 genes),
and AI (37% of 6579 probe sets within a species) consistent with
previous reports on other platforms (McIntyre et al. 2006; Wayne
et al. 2007; Telonis-Scott et al. 2008; Fontanillas et al. 2010).
MATERIALS AND METHODS
The chip has 2,424,414 informative features, covering four types of
probes: SNP probes (n ¼ 1,442,832; 60,118 probe sets); 39 expression
probes (n ¼ 262,766; 18,769 probe sets); exon probes (n ¼ 699,865;
61,919 probe sets); and control probes (16,943 GC band controls; 2008
hybridization and labeling controls; Figure 1). The 39 expression
probes consist of all perfect-match (PM) probes from the Affymetrix
GeneChip Drosophila Genome 2.0 array (900531, 900532, and
900533). The exon probe sets provide measurements of expression
from each individual exon, allowing controls for signal fluctuation
caused by 59 bias in expression assays, as well as measurement of
alternative exon usage. The exon probes consist of all Affymetrix
Drosophila Tiling 2.0 Array (901021) probes that map uniquely to
exonic regions (FlyBase R5.11 August 2008) at the time of chip design.
Overlapping exons with alternative start/end sites in the same geno-
mic region were combined into a single exonic region. The majority of
exonic regions contain a single exon. (For simplicity, exonic regions
are referred to simply as exons throughout this article.) Each exon
corresponds to a unique probe set. The 39 expression probes and exon
probes on this custom chip were designed by Affymetrix from
D. melanogaster sequences. The probe sets have been used for other
Drosophila species (i.e., Kopp et al. 2008; Graze et al. 2009; Dworkin
and Jones 2009; Lu et al. 2010). Using these probe sets allows direct
comparisons to existing literature and straightforward quality control.
As each probe set has multiple probes, the impact of divergence is
likely to be minimal on summary measures of expression. However,
investigators comparing among species should consider filtering in-
dividual probes. The SNP module was designed for estimating AI.
There were three main steps in this design: SNP identification, SNP
quality assessment, and probe selection.
SNP identification: Alignment sets were created from multiple
sequence sources, including FlyBase R5.4 exons (n ¼ 68,536), six
D. simulans strain genomes for Drosophila Population Genomics Pro-
ject (DPGP, http://www.dpgp.org, Begun et al. 2007), and all D. sim-
ulans sequences (343,420) from GenBank (Benson et al. 2005) that
were not annotated as “whole genome.” In DPGP, D. simulans
genomes, except for the heterochromatic regions, were assembled
against the FlyBase R4.2 D. melanogaster genome. Exons from FlyBase
R5.4 were BLAST (Altschul et al. 1990) aligned to the DPGP genomes
and GenBank sequences. There were 325 exons with only GenBank
sequence, 62,161 with only DPGP sequence, 3163 with both GenBank
and DPGP sequence, and 2887 for which no sequence was available.
For each FlyBase R5.4 exon, its genome location in D. melanogaster
R4.2 genome was determined by BLAST exons that matched more
than one location; those located on chromosomes four or U were
excluded (n ¼ 1912). All matching sequences for each exon were
aligned using ClustalW (Thompson et al. 1994) to create a multiple
sequence alignment at the exon’s genome position. All SNPs, regard-
less of location in the exon, were identified from the multiple sequence
SNP quality assessment: A design window, which consisted of 217
bases upstream and 17 bases downstream from each SNP (Figure 2)
Figure 1 Probe design. A total of 2,424,414 probes
were printed on the chip. They are of four types: SNP
probes (n ¼ 1,442,832; 60,118 probe sets), 39 expres-
sion probes (n ¼ 262,766; 18,769 probe sets), exon
probes (n ¼ 699,865; 61,919 probe sets), and control
probes (not shown). The 39 expression probes consist of
all perfect-match (PM) probes from the Affymetrix Gene-
Chip Drosophila Genome 2.0 array. An example of a 39
expression probe set is shown in navy. The exon probes
consist of all Affymetrix Drosophila Tiling 2.0 Array
probes that map uniquely to exonic regions annotated
in FlyBase R5.11 (August 2008). Exon probe sets within
the example gene are shown in light blue. SNP probes
are custom made. The SNP probes corresponding to a
single SNP site base are shown in dark blue (matching the
forward strand) and blue (matching the reverse strand).
| Y. Yang et al.
was the basis of SNP quality assessment. A SNP locus supported by
fewer than five sequences was discarded. SNPs were also discarded
when the design window mapped to multiple places in the genome or
when more than one SNP occurred in the design window. These
criteria identified 589,915 SNPs, of which 196,345 were biallelic. Only
biallelic SNPs were considered further. There were 558 exons for
which SNP data were identified from GenBank alone, 51,418 exons
for which SNP data were identified from DPGP alone, and 2992
identified from both, resulting in a total of 54,968 exons with SNPs
present; in other words, 81% coverage of the entire FlyBase R5.4
transcriptome (68,536 exons).
Probe selection: For each SNP, 24 probes were designed, with the
SNP at the 0, +4, and 24 positions from the probe center, for the
forward and reverse strands, and with each possible base (A, C, G, and
T) at the SNP site. Probe hybridization quality was predicted by an
Affymetrix internal scoring algorithm that takes into account Tm,
secondary structure, and previous empirical observations. If a probe
contained a homopolymer run or could not be synthesized or if one
third or more probes had poor predicted hybridization, the probe set
was eliminated. For genes with seven or fewer SNPs, all SNPs were
selected. If a gene had more than seven SNPs, additional probe sets
were selected at random (n = 610) to fill the chip.
In sum, 60,118 custom SNP probe sets representing 11,929
genes (Figure 3) were included on the chip. The mean number
of probe sets per gene was 4.4. The majority (8013 genes) had
more than 3 probe sets. The chip library files are available at
http://bioinformatics.ufl.edu/McIntyre_Lab/ASE. Probe sequen-
ces and chip annotation can be found at Gene Expression Om-
nibus (GEO) using accession ID GPL11273.
Verifying the experiment fly materials
Experimental design:Two different isogenic strains of D. simulans,
st e and C167.4, and their male and female progeny were used as the
basis for the verification study. Three replicates of RNA from female
and male progeny of the cross st e · C167.4 were assayed for six RNA
samples. In addition, DNA was used as a control for estimating AI
(Wittkopp et al. 2004; Wittkopp et al. 2008; Degner et al. 2009;
McManus et al. 2010). Three replicate gDNA samples were prepared
for female st e, female C167.4, and the female F1progeny of the cross
st e · C167.4, for nine gDNA samples.
Sample collection: Flies were reared in incubators (25?, 12:12 hr
light/dark cycle) on a standard dextrose medium. Isogenic strains
of D. simulans (C167.4, BDSC 4736; st e isogenic, DSSC 14021-
0251.041 inbred .20 generations) were used. For each of three cross
genotypes (C167.4, st e, and C167.4 · st e), 20 virgin females were
crossed to 5 males. Female and male progeny were collected on
consecutive days (under CO2) and aged from 5 to 7.5 days in single
sex vials. Flies were then flash frozen in liquid nitrogen (without
anesthesia) in a 2.5 hr window (4–6:30 PM). For RNA samples, two
sets of 20 flies (subsamples) were collected for each replicate from
multiple cross vials. No vials were used for more than one replicate.
Sample processing: Flies were freeze dried at 220? overnight prior to
homogenization. Dried flies were ground to a fine powder using
a GenoGrinder (maximum, 3 min, repeated twice). Trizol (1 ml)
was added to each homogenized sample and mixed thoroughly in
the GenoGrinder (maximum, 3 min). Samples were transferred to
a new tube, 1 ml linear acrylamide was added to each, and then
samples were incubated at room temperature for 5 min. RNA was
extracted using a standard Trizol extraction protocol: phase separation
using 0.1 vol BCP, RNA precipitation with isopropanol, 70% ethanol
wash, and resuspension in 80 ml DEPC H2O. Concentration was
measured using a NanoDrop, and up to 30 mg RNA per sample
was treated with DNase I in 100 ml reaction volumes for 30 min at
37? (reaction mix: 4 U Cloned DNase I TaKaRa 2220A, 80 U Promega
Recombinant RNasin N2515, in 1· TaKaRa Cloned DNase I Buffer
II). Samples were cleaned prior to concentration using the Qiagen
RNeasy Mini Kit (Cat. #74104) following the manufacturer’s standard
protocol with 30 ml DEPC H2O elutions (run through the column
twice). RNA quality was examined using BioAnalyzer RNA 6000
Nano chips, and all samples were found to be of good quality. Geno-
mic DNA was isolated from 35 to 40 flash frozen females using the
AllPrep Mini Kit (Qiagen) following standard manufacturer’s proto-
cols. Samples were concentrated by standard ethanol precipitation and
resuspended in 31 ml DEPC H2O.
Fragmentation, labeling, and array hybridization: Target materials
were prepared for array hybridization using the recommended
Affymetrix kits following the no amplification protocol of GeneChip©
WT Double-Stranded Target Assay Manual (DNA samples started
Figure 2 SNP probe design windows. For each SNP site, there are
four sets of probes, one for each SNP site base. The SNP base is
designed at three different positions of the probes: middle, shifted
four bases upstream, or shifted four bases downstream. Each SNP
probe set contains 24 probes, which can be classified based on alleles
as PM1 (n ¼ 6), PM2 (n ¼ 6), or MM (n ¼ 12), for a total of 24 probes
per probe set. A SNP probe set has a 35-base design window, with
sequences of 217 bases upstream and 17 bases downstream from the
SNP. If there were fewer than five sequences supporting a SNP, the
SNP was discarded. If more than one SNP occurred in the design
window, then the alignment was considered suspect, and the SNP
was not included among those printed on the array. Only biallelic
SNPs that were unique in their design window and supported by five
or greater sequences in the multiple alignment were considered.
Volume 1November 2011| A Custom Array for Abundance, Isoforms, and Alleles|
from Procedure D forward) for single Tiling Arrays. Briefly, 10 mg of
total RNA was concentrated to 8 ml in DEPC H2O followed by first-
and second-strand cDNA synthesis using the WT Double-Stranded
DNA Synthesis Kit (P/N 900813). Per the GeneChip Sample Cleanup
Manual (P/N 900371), 7.5 mg of dsDNA was fragmented. For
each DNA sample, 7.5 mg of gDNA was fragmented to between 25
and 200 bp with 0.02 U/mg DNase I (Takara Cloned DNase I, 2 U/ml)
in a 40 ml reaction with 4 ml reaction buffer (10· reaction buffer: 100
mM Tris-acetate, 100 mM magnesium acetate, 100 mM potassium
acetate) and 0.8 ml BSA (10 mg/ml). Reactions were incubated 16
minutes at 37? and heat killed at 99? for 15 minutes. Fragment size
was checked by agarose gel electrophoresis. Fragmented cDNA and
gDNA targets were labeled using WT Double-Stranded DNA Terminal
Labeling Kit (P/N 900812). The prepared target samples were hybrid-
ized using the Hybridization, Wash, and Stain Kit (P/N 900720) fol-
lowing the manufacturer’s protocol (FS450_0001) for the Fluidics
Station 450 with protocol. Arrays were scanned using an Affymetrix
7G scanner. The GEO accession for the array data is GSE31750.
Signals were extracted from the scans using the apt-cel-extract
program of the Affymetrix Power Tools (version 1.10.2) suite. GC
bin control probes provide an estimate of nonspecific hybridization
(Affymetrix 2005) and help to assess the overall quality of the
hybridization. A GC bin control is a standard Affymetrix control
based upon the number of G/C bases (from 3 to 24) in the 25 mer
probe. None of the GC bin control probes align to the D. melanogaster
or D. simulans reference genomes. Individual probes were classified
according to their GC content and matched to the corresponding GC
bin controls. A probe was considered detected when signal strength
was higher than the median intensity of the corresponding GC band
controls. Detection above background (DABG) was calculated at the
individual probe level. The overall intensity of the array was evaluated
at the individual probe level. To correct for the background noise
and to normalize the probe signals, each probe was classified into
a GC bin and the 5 percentile signal for that GC bin was subtracted
from the probe signal. Yi, the signal for probe set i, is estimated as:
jðXij2GCjÞ=Niþ 100Þ. Xijis the intensity for probe j in
probe set i and GCjis the average intensity for control probes in the
corresponding GC bin. Niis the number of probes in probe set i. Chip
verification was analyzed first for the overall hybridization quality,
then for each module on the chip (39 expression module, exon mod-
ule, and SNP module).
General quality control
The distribution of the overall signal across all modules was compared
using kernel density estimates for each slide separately (Silverman
1986), with the goal of identifying any slide with an unusual distri-
bution. Similar marginal distributions of kernel density would be
expected for one sample type. Principle component analysis (PCA)
(Johnson and Wichern 1992) was carried out to determine whether
there was any pattern or grouping to the data.
To verify the veracity of probe set estimates of 39 expression, we
compared the signal from probe sets for the well-known sex-biased
genes (Bownes 1994; Wolfner 1997): Yp (Yp1, Yp2, Yp3) and Acp
(Acp29AB, Acp32CD, Acp36DE, Acp53C14a, Acp53C14b, Acp53C14c,
Acp62F, Acp76A). Consistency of estimation of gene expression
across modules was also examined using Bland-Altman plots (Bland
and Altman 1986; Bland and Altman 1988; Dudoit et al. 2002;
McIntyre et al. 2011), in which the exon module and SNP module
were plotted against the 39 expression module.
The feature size of this array is smaller (5 micron) than is the
Affymetrix GeneChip Drosophila Genome 2.0 array (11 micron). Al-
though the PM probes are identical for these two chips, feature size
may have an impact on differential expression (Dandy et al. 2007;
Ammar et al. 2009). This raises the concern of potential loss of sen-
sitivity. To evaluate the performance of the 39 expression and exon
modules, we compared expression for RNA samples between the two
sexes of the F1progeny. Sex-biased expression is well described for
Drosophila (Bownes 1994; Jin et al. 2001; Parisi et al. 2003; Ranz et al.
2003; McIntyre et al. 2006; Telonis-Scott et al. 2008).
The fixed effects model,
Yij¼ m þ siþ eij
was fit for each probe set in the 39expression and exon modules,
where Yijis the signal for probe set i, sample j is as described above,
m is the overall mean, siis the fixed effect of sex, and eijis the
random error. The null hypothesis that male and female sexes had
equal expression levels was tested using an F-test (Neter et al. 1990).
All probes in a given probe set were used. As only one genotype is
considered, any polymorphisms between the genotype used and the
probe will be the same between the two sexes of the same genotype.
Results were corrected for multiple testing using False Discovery
Rate (FDR) (Benjamini and Hochberg 1995; Verhoeven et al. 2005).
Where multiple probe sets matched the same genes, the agreement
between the exon probe sets and the 39 expression probe sets were
examined for agreement in detecting sex bias using Kappa statistics
(Fleiss 1981) and McNemar’s test (Johnson and Wichern 1992).
There is a sex bias in isoform usage in Drosophila (Kwan et al.
2008; Telonis-Scott et al. 2008; McIntyre et al. 2006). The use of
alternative transcript isoforms between the two sexes can be detected
from the measurements taken by the exon module. A probe set rep-
resents a constitutive exon for a gene (included in all annotated iso-
forms) or an alternative exon (included in a subset of known
isoforms). Inferences can become ambiguous when probe set annota-
tions correspond to exon regions located in overlapping regions of
multiple gene models. Probe sets mapping to more than one gene or
to the ambiguous regions of overlapping exons were excluded from
analysis (n = 2611). The model
Figure 3 Distribution of SNP probe sets per gene. A total of 60,118
probe sets, representing 11,929 genes, were selected for the SNP
module for the custom chip. The number of genes (Y axis) with a given
number of corresponding SNP probe sets (X axis) is shown. Most
genes are represented on the array by one to five SNP probe sets.
| Y. Yang et al.
Yij¼ m þ xiþ sjþ xsijþ eij
where x is the fixed effect of exon type and s is the fixed effect of sex
was fit. Probe sets from multiple constitutive exons were grouped as
one exon type, whereas probe sets representing alternative exons
were each considered a different exon type. The variance was esti-
mated separately for each sex. The significance of the interaction (a
test for alternative isoform usage McIntyre et al. 2006) was tested
using an F test, followed by FDR correction.
SNP calling and genotyping accuracy: By design, there are DPGP/
GenBank sequences for all 60,118 probe sets in the SNP set, from
which biallelic SNPs were defined and used for the chip design. SNP
alleles were verified using Illumina genome resequencing data for the
C167.4 and st e strains (GEO accession SRP005952) of D. simulans.
D. simulans C167.4 sequence data were obtained from male head
RNA libraries sequenced on multiple lanes with Illumina paired end
procedures and chemistry (Celniker et al. 2009; McIntyre et al. 2011).
D. simulans st e sequences were from genomic DNA extracted from
adult st e D. simulans females. Average coverage was 30·. Reads were
aligned to updated reference genomes (R. Graze et al., unpublished
data) using Bowtie (Langmead et al. 2009) and LAST (Frith et al.
2010). Alignments were converted to pileup format using SAMtools
(Li et al. 2009). SNP bases were identified from the pileup alignments
and compared with alleles identified from DPGP/GenBank. The bases
identified from C167.4 resequencing were also compared with the
DPGP genome sequences for the C167.4 strain.
Analysis of AI
To verify the chip’s capacity to identify differences in AI, a subset of
SNP probe sets unambiguous for the two alleles from the design and
confirmed by the C167.4 resequencing and st e resequencing (where
the F1is heterozygous) were selected. The model,
Yijk¼ m þ siþ tjþ eijk
was fit for probe sets in this subset using the nine F1arrays (six RNA,
three DNA). Yijis the normalized signal value for the ithsex, jth
treatment, and the kthreplicate. The treatment groups were defined
by combinations of nucleic acid (DNA/RNA) and allele (PM1, PM2,
and MM) for a total of j = 1...6 levels and k = 1...3 replicates. AI
was examined by testing the difference in hybridization intensity
between PM1 and PM2 in the RNA, compared with the difference
in the DNA. An F test for this contrast was performed, and the result
was corrected for multiple testing using FDR.
The power of detection of AI effects may differ between the sexes
due to sex bias in gene expression. There may also be sex-specific
differences in AI. Both phenomena would result in a difference in
detection of AI between the sexes. Unfortunately, the power for the
test of an interaction was low. The AI was also analyzed considering
the female and male data separately so that any differences between
the sexes in the results might be apparent.
Quality control evaluations showed that the three C167.4 parental
DNA hybridizations had overall weaker signals and that the kernel
density distribution was markedly different from all of the other chips.
The DABG was only 70% for these hybridizations (Figure 4A) com-
pared with ?90% for other DNA samples. The remaining chips
showed no obvious problems with hybridization. All modules (39
expression, exon, and SNP) had similar proportions of probes
detected above the median of the GC band control signals (Table
1). The proportions were ?72% for RNA samples and ?90% for
DNA samples. The distribution of signal values across all modules
Figure 4 Quality control analyses of the normalized data. (A) The
proportion of probes detected above background (DABG) is reported
for all probes sets of each sample: C167.4 parental DNA, st e parental
DNA, and DNA and RNA of the F1genotype. DNA samples are shown
in dark gray. RNA samples are shown in light gray. The Y axis is the
overall percentage of DABG. Probes were classified according to their
GC content and matched to the GC band controls of the correspond-
ing %GC bin. A probe was considered detected when signal strength
was higher than the median intensity of the corresponding GC band
controls. The three C167.4 parental DNA hybridizations had lower
DABG compared with the other two genotypes of DNA samples. (B)
Box plot for probe intensity classified by genotype and nucleic acid.
DNA samples are shown in dark gray. RNA samples are shown in light
gray. The Y axis is the normalized signal. Probes were classified
according to their GC content and matched to the GC band controls
of the corresponding %GC bin. The five percentile signal for that GC
bin was subtracted from the probe for background correction. The
corrected signals were then log-transformed. The three C167.4 paren-
tal DNA hybridizations had overall weaker signals.
Volume 1November 2011| A Custom Array for Abundance, Isoforms, and Alleles|
was similar for all RNA hybridizations and differed from DNA
hybridizations (Figure 4B). PCA identified no other hybridization
anomalies. Both sexes had similar hybridization patterns (supporting
information, Figure S1). The normalized signal intensities of the exon
module probe sets and expression module probe sets for Acp and Yp
genes gave the expected results (Figure 5). The average intensity for
each gene was consistent between modules (Figure S2).
To test the smaller format features of the 39 expression probe sets,
we analyzed the effect of sex on expression level. Previous studies in
Drosophila (Bownes 1994; Jin et al. 2001; Parisi et al. 2003; Ranz et al.
2003; McIntyre et al. 2006; Telonis-Scott et al. 2008) have all found
sex bias in overall expression. Analysis of differential expression on
the 39 expression module revealed a strong effect of sex on gene
expression. Three different significance levels (FDR , 0.05, FDR ,
0.1, and FDR , 0.2) are reported (Table 2). Raw P values and adjusted
FDR P values are in Table S1. Among all sex-biased probe sets at
FDR , 0.1, 2216 had higher expression in males and 4140 had higher
expression in females. Sex bias of expression was similarly analyzed
for exon probe sets corresponding to single exons that exist in all
transcripts of a gene (constitutive) (n = 47,122; Table 2; Table S3).
The results for exon probe sets were compared with the 39 expression
probe sets corresponding to the same genes. There were 2091 genes
with probe sets corresponding to exons contained in all transcripts,
which could be easily matched to a single probe set in the 39 expres-
sion module. The majority of genes showed similar sex bias between
the 39 expression and exon module, and simple agreement was high
(1720, 82%). The Kappa statistic was 0.63 between the two modules,
indicating good agreement. There was no apparent asymmetry in
detection, with 106 genes detected by the exon module alone and
265 genes detected by the 39 expression module alone. The McNe-
mar’s test statistics was 68.1429 with P value smaller than 0.0001.
There were 164 genes that showed a significant interaction between
exon type and sex; these were considered as showing putative isoform-
specific sex bias (FDR , 0.1; Table S2).
n Table 1 Proportions of probes detected above the median GC band control
Exon module DABG
Expression module DABG
SNP module DABG
The average proportion of probes detected above background (DABG) for genotype, nucleic acid, and sex. An individual probe was detected when signal strength
was higher than the median intensity of the corresponding GC band control. Average DABG was calculated for each individual module and for the overall slide. The
individual modules of expression, exon, and SNP, as well as the entire slide, had similar proportions of probes detected above the median GC band control. The
distribution of signal values across all modules was similar for all RNA hybridizations and differed from DNA hybridizations. The three C167.4 parental DNA
hybridizations had lower DABG compared with the other two genotypes of DNA samples.
Figure 5 Expression for known sex-specific genes in female and male RNA samples. The Y axis is the normalized signal. A value around or lower
than 3 is close to the background intensity and, therefore, should be considered as not detected. Female samples are shown in red. Male samples
are shown in blue. (A) The mean signals of all probe sets for each Acp gene. (B) The expression of individual probe sets designed for Acp genes.
(C) The mean signals of all probe sets for each Yp gene. (D) The expression of an individual probe set that was designed for Yp genes. The
directions of sex bias are as expected (Acps are male-specific genes, and Yps are female-specific genes). Individual probe sets for the same gene
| Y. Yang et al.
Verifying the SNP module
The D. simulans sequences used to design the SNP probes were se-
quenced as part of the DPGP. The sequencing strategy of DPGP was
to sequence one line at ?4· coverage and six additional lines (in-
cluding C167.4) at 1·. The confidence for SNP calls, therefore, varied
depending on quality and depth. There were 60,118 probe sets, and of
these, 42,978 had SNP base information for C167.4 from DPGP,
35,379 had additional data available from Illumina sequencing of
C167.4 RNA, and 49,758 had additional data from Illumina sequenc-
ing of st e DNA.
The concordance of C167.4 SNP base calls from the ?1· DPGP
C167.4 strain genome sequence used for the chip design and the
resequencing was 66.72%, significantly larger than expected by chance.
This rate did not affect the quality of the SNP probe sets, as C167.4
was only one of the seven lines in DPGP that were used for our chip
design. The concordance between the resequencing bases and the
alleles used in the design was impressive. The C167.4 RNA-Seq base
agreed with one of the two alleles identified in the design 99.58% of
the time. Agreement between the st e DNA-Seq bases and the two
alleles in the design was 99.75%.
Next, the concordance between the resequencing and hybridiza-
tion was examined by comparing resequencing SNP base calls to the
probe bases ranked by the strength of their hybridization signals. The
comparison was carried out separately for cases where the resequenc-
ing bases were the same for C167.4 and st e (homozygous F1) or
where they were different (heterozygous F1). For n = 13,573 SNP
probe sets homozygous at the SNP site in the C167.4/st e genotype,
the ranked hybridization intensities of probes corresponding to each
base (within a given SNP probe set) was compared with the predicted
genotype at the SNP base (Table 3). A SNP probe set was included in
this comparison when the common SNP allele call for C167.4 and st e
strains corresponded to one of the alleles in the chip design, and the
base was also the same in the DPGP sequence for the C167.4 strain.
For example, if the SNP allele in both strains is A (with respect to the
forward strand), probes corresponding to targets with A at the SNP
position are expected to show increased hybridization intensity rela-
tive to the signal for the T, G, and C probes for all genotypes tested.
The percentage of probe sets where the probes corresponding to the
target SNP allele show the highest intensity is reported overall and
separately for each base (Table 3). The concordance between the
resequencing SNP and the C167.4 DNA arrays was significantly lower
than other arrays, likely caused by the weaker signal intensity of the
C167.4 DNA arrays. Similarly, the ranked hybridization intensities of
probes corresponding to each base (within a given SNP probe set) was
compared with the predicted genotype at the SNP base for n = 2769
SNP probe sets heterozygous at the SNP site in the C167.4/st e ge-
notype (Table S4). Our observed concordance is striking, given the
small number of genotypes used in this experiment. A previous study
(Borevitz et al. 2007) using arrays for detecting sequence polymor-
phisms reported a similar error rate. Larger experiments with more
samples can reduce the error rate (Edenberg et al. 2005; Rabbee and
Speed 2006). In comparison, a short-read sequencing experiment
requires more than 200 reads unambiguously mapped to the gene
for each gene to achieve a similar result (Fontanillas et al. 2010).
The hybridization signals were compared with the two alleles
(PM1 and PM2) used in the design. The concordances between the
two highest ranked probe bases and the PM1 and PM2 bases were
high: 96.22% for C167.4 DNA chips and 98.77% for st e DNA chips.
For hybrid F1’s, the concordance percentage was 98.62% for DNA
chips and 97.53% for RNA chips. To determine whether the pattern
of hybridization could be used to predict genotype, we performed
a linear discriminate analysis (Johnson and Wichern 1992) for several
arbitrarily selected probe sets heterozygous for the SNP base in the F1
hybrid. All that we examined showed visual separation of the expres-
sion patterns (Figure 6). These comparisons confirmed that the
hybridizations performed as expected.
n Table 2 Tests for the effect of sex from the 39 expression and exon probe modules at multiple FDR levels
FDR , 0.05
FDR , 0.1
FDR , 0.2
39 Expression module (n ¼ 18,769)
Exon module (n ¼ 47,122)
The probe sets in the 39 expression module and the exon module were tested for sex-biased expression. The number of significant probe sets and the percentage of
significant probe sets over all probe sets within a module are shown. Results from different significance thresholds (FDR , 0.05, 0.1, and 0.2) all indicate a strong sex
effect measured by both the expression module and the exon module.
n Table 3 Rank of hybridization signal corresponds to the expectation based on sequence information (homozygous genotypes)
Considered were probe sets where 1) SNP allele calls for the C167.4 and st e strains correspond to the PM1 and PM2 alleles in the chip design; 2) the C167.4 base
is concordant between the resequencing data and the DPGP sequence for the C167.4 strain; and 3) the C167.4/st e genotype is homozygous for the SNP site
(n ¼ 13,573). The signal from each base was estimated as the average of the probes representing that base. For each probe set, the four bases were ranked according
to signal, and the base with the greatest hybridization signal was compared with the known base. The percentage of probe sets for which the base corresponding to
the top-ranked hybridization intensity was the known allele was calculated. Percentages are reported considering all SNP bases and separately for A, C, G, and
T alleles. The concordance between the resequencing SNP and the C167.4 DNA arrays was significantly lower than other arrays, likely due to the weaker signal
intensity of the C167.4 DNA arrays.
Volume 1November 2011| A Custom Array for Abundance, Isoforms, and Alleles|
There were 33,914 unambiguous probe sets with sequence informa-
tion for both the st e and C167.4 resequencing experiments at the
SNP site. The SNP base was the same for the parental two lines for
74.78% of these probe sets, which were classified as homozygous for
the F1genotypes (n = 25,362). The rest of the probe sets (8552) had
heterozygous F1genotypes. The 6579 autosomal probe sets were an-
alyzed for AI on the combined data from both sexes and for each sex
separately (Table 4; Table S5).
Our custom platform performs similarly to previous array platforms
with a larger feature size (Jin et al. 2001; Ranz et al. 2003; McIntyre
et al. 2006; Wayne et al. 2007). McIntyre et al. (2006) analyzed 10,014
transcripts in eight lines of D. melanogaster and identified 5221 sex-
biased transcripts at FDR 0.05 (56% male bias and 44% female bias).
The overall sex effect for eight genotypes was 53%. Similarly, on
a study using nine D. melanogaster lines, Wayne et al. (2007) reported
7617 out of 9312 genes with sexually dimorphic expression (4070 male
bias and 3547 female bias). These previous studies used multiple
genotypes of D. melanogaster. When the data for Wayne et al.
(2007) were reanalyzed for each genotype separately, the percentage
of sex-biased genes ranged from 1.29 to 40.09%. For the genotype
considered in this study, 31% of genes showed a significant sex effect,
close to the upper end of the range. A slight excess of genes with
increased expression in females was also observed in this analysis, as is
seen in previous analyses (Ranz et al. 2003). These findings are con-
sistent with findings from arrays with a larger feature size.
Two previous studies using array designs (McIntyre et al. 2006;
Telonis-Scott et al. 2008) found significant sex differences in alterna-
tive exon usage for many genes. For the single genotype examined
here, approximately 5.6% of the genes examined showed evidence of
sex-specific isoform expression. For four genes that are components of
the sex determination pathway with previously reported sex-specific
splicing in adults (tra2, Sxl, dsx, and fru), at least one exon shows
evidence of sex bias.
This is the first genome-wide study of allele-specific expression
variation within D. simulans. Although only one genotype was used,
significant differences in AI were detected for 37% of probe sets exam-
ined that contained heterozygous SNP loci. Other work using a priori
plots of three genotypes: AA,
AC, and CC. Different geno-
types had hybridization patterns
that are visually separable by
linear discriminant (LD) analysis.
Each genotype is a different
6 Linear discriminant
n Table 4 Allele imbalance overall and separated by sex (n ¼ 6,579)
FDR , 0.05
FDR , 0.1
FDR , 0.2
AI was tested for male and female samples alone and combined. Results from
different significance cutoffs (FDR , 0.05, 0.1, and 0.2) are shown. The numbers
of probe sets with a significant AI effect are reported. There are large propor-
tions of genes that have significant AI in male samples, female samples, and
combined (male/female) samples.
| Y. Yang et al.
selected genes found almost 67% of the genes tested showed evidence for
AI within species (Wittkopp et al. 2008). This chip can be used to detect
allele-specific variation in expression within species. Large differences
between males and females were detected in the number of probe sets
that showed significant differences in AI. It is currently unclear whether
this result is explained by differences in AI between males and females
or by sex bias in overall expression making power for detection uneven.
As in microarray studies, to adequately assess ASE for a particular
transcript using RNA-Seq, there must be adequate coverage for both
alleles of that particular gene. For samples from the same organism and
tissue, detection of transcription for a particular exon in four gigabases
of RNA-Seq data are 57% (R. Graze et al., unpublished data), whereas
detection is 72% for tiling arrays (Graze et al. 2009). Initial studies of
AI examined how many informative reads were needed per gene for
estimation of allelic frequencies (Fontanillas et al. 2010). This study
suggests that average depth of coverage needed is quite large if most
genes are to be evaluated. The actual coverage needed depends on
specific assumptions and the number varies, but it is often in excess
of 100·. Other examinations of RNA-Seq find that a minimum average
depth of five reads per nucleotide are needed for estimation of expres-
sion (McIntyre et al. 2011). One lane of a GAIIX provides sufficient
reads at a coverage of 5· to assess ?30% of the transcriptome (McIn-
tyre et al. 2011). Arrays still provide a cost effective way of assessing
transcription for the whole genome (Malone and Oliver 2011).
Detailed studies within species that examine AI variation genome-
wide and identify the impact of sex on this variation are needed to
understand the true extent of cis regulatory variation within species in
Drosophila. This array is a good tool for such studies because it will
allow the overall and allele-specific components of expression varia-
tion to be examined in a single experiment on a single platform for
many more genes than has previously been possible.
This work is supported by National Institutes of Health grants
5R01GM077618-A5 and 5R01GM077618-S1.
Affymetrix, Inc. 2005
Ahmadian, A., B. Gharizadeh, A. C. Gustafsson, F. Sterky, P. Nyrén et al.,
2000 Single-nucleotide polymorphism analysis by pyrosequencing.
Anal. Biochem. 280: 103–110.
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman,
1990Basic local alignment search tool. J. Mol. Biol. 215: 403–410.
Ammar, R., A. M. Smith, L. E. Heisler, G. Giaever, and C. Nislow, 2009
A comparative analysis of DNA barcode microarray feature size.
BMC Genomics 10: 471.
Begun, D., A. Holloway, K. Stevens, L. Hillier, Y.-P. Poh et al.,
2007 Population genomics: whole-genome analysis of polymorphism
and divergence in Drosophila simulans. PLoS Biol. 5: e310.
Benjamini, Y., and Y. Hochberg, 1995
a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57:
Benson, D. A., I. Karsch-Mizrachi, D. J. Lipman, J. Ostell, and D. L. Wheeler,
2005 GenBank. Nucleic Acids Res. 33: D34–D38.
Bland, J. M., and D. G. Altman, 1986
ment between two methods of clinical measurement. Lancet 1: 307–310.
Bland, J. M., and D. G. Altman, 1988
books, software and manuals. Int. J. Epidemiol. 17: 201–203.
Borevitz, J. O., S. P. Hazen, T. P. Michael, G. P. Morris, and I. R. Baxter et al.,
2007 Genome-wide patterns of single-feature polymorphism in Arabi-
dopsis thaliana. Proc. Natl. Acad. Sci. USA 104: 12057–12062.
Exon array background correction. Available at:
Controlling the false discovery rate:
Statistical methods for assessing agree-
Misleading statistics: errors in text-
Bownes, M., 1994
sex differentiation genes in Drosophila melanogaster. Bioessays 16:
Celniker, S. E., L. A. L. Dillon, M. B. Gerstein, K. C. Gunsalus, S. Henikoff
et al., 2009 Unlocking the secrets of the genome. Nature 459: 927–930.
Dandy, D. S., P. Wu, and D. W. Grainger, 2007
ences nucleic acid surface capture in DNA microarrays. Proc. Natl. Acad.
Sci. USA 104: 8223–8228.
Degner, J. F., J. C. Marioni, A. A. Pai, J. K. Pickrell, and E. Nkadori et al.,
2009 Effect of read-mapping biases on detecting allele-specific expres-
sion from RNA-sequencing data. Bioinformatics 25: 3207–3212.
de la Chapelle, A., 2009 Genetic predisposition to human disease: allele-
specific expression and low-penetrance regulatory loci. Oncogene 28:
Dudoit, S., Y. H. Yang, M. J. Callow, and T. P. Speed, 2002
methods for identifying differentially expressed genes in replicated cDNA
microarray experiments. Statist. Sinica 12: 111–139.
Dworkin, I., and C. D. Jones, 2009 Genetic changes accompanying the evo-
lution of host specialization in Drosophila sechellia. Genetics 181: 721–736.
Edenberg, H., L. Bierut, P. Boyce, M. Cao, S. Cawley, et al., 2005
of the data from the Collaborative Study on the Genetics of Alcoholism
(COGA) and single-nucleotide polymorphism genotyping for Genetic
Analysis Workshop 14. BMC Genet. 6(Suppl. 1):S2.
Emerson, J. J., L.-C. Hsieh, H.-M. Sung, T.-Y. Wang, C.-J. Huang et al.,
2010 Natural selection on cis and trans regulation in yeasts. Genome Res.
Fleiss, J., 1981Statistical Methods for Rates and Proportions. Wiley,
FlyBase, 2008A database of Drosophila genes and genomes.
Fontanillas, P., C. R. Landry, P. J. Wittkopp, C. Russ, J. D. Gruber et al.,
2010Key considerations for measuring allelic expression on a genomic
scale using high-throughput sequencing. Mol. Ecol. 19: 212–227.
Frith, M. C., R. Wan, and P. Horton, 2010
data into alignment improves DNA read mapping. Nucleic Acids Res. 38:
Graze, R. M., L. M. McIntyre, B. J. Main, M. L. Wayne, and S. V. Nuzhdin,
2009Regulatory divergence in Drosophila melanogaster and D. simu-
lans, a genome-wide analysis of allele-specific expression. Genetics. 183:
Guo, M., S. Yang, M. Rupe, B. Hu, D. Bickel et al., 2008
allele-specific expression analysis using Massively Parallel Signature Se-
quencing (MPSS) reveals cis- and trans-effects on gene expression in
maize hybrid meristem tissue. Plant Mol. Biol. 66: 551–563.
Jin, W., R. M. Riley, R. D. Wolfinger, K. P. White, G. Passador-Gurgel et al.,
2001The contributions of sex, genotype and age to transcriptional
variance in Drosophila melanogaster. Nat. Genet. 29: 389–395.
Johnson, A. D., D. Wang, and W. Sadee, 2005
gene regulation and mRNA processing: broad implications for pharma-
cogenetics. Pharmacol. Ther. 106: 19–38.
Johnson, J. M., J. Castle, P. Garrett-Engele, Z. Kan, P. M. Loerch et al.,
2003Genome-wide survey of human alternative pre-mRNA splicing
with exon junction microarrays. Science 302: 2141–2144.
Johnson, R. A., and D. W. Wichern, 1992
Analysis. Prentice Hall.
Kopp, A., O. Barmina, A. M. Hamilton, L. Higgins, and L. M. McIntyre et al.,
2008Evolution of gene expression in the drosophila olfactory system.
Mol. Biol. Evol. 25: 1081–1092.
Kwan, T., D. Benovoy, C. Dias, S. Gurd, C. Provencher et al.,
2008 Genome-wide analysis of transcript isoform variation in humans.
Nat. Genet. 40: 225–231.
Langmead, B., C. Trapnell, M. Pop, and S. Salzberg, 2009
memory-efficient alignment of short DNA sequences to the human
genome. Genome Biol. 10: R25.
Li, H., B. Handsaker, A. Wysoker, T. Fennell, J. Ruan et al., 2009
Sequence Alignment/Map (SAM) Format and SAMtools. Bioinformatics
The regulation of the yolk protein genes, a family of
Array feature size influ-
Incorporating sequence quality
Applied Multivariate Statistical
Volume 1November 2011|A Custom Array for Abundance, Isoforms, and Alleles|
Lo, H. S., Z. Wang, Y. Hu, H. H. Yang, S. Gere et al., 2003 Download full-text
in gene expression is common in the human genome. Genome Res. 13:
Lu, X., J. A. Shapiro, C.-T. Ting, Y. Li, C. Li, et al., 2010
expression of X-linked versus autosomal genes associated with hybrid
male sterility. Genome Res. 20: 1097–1102.
Malone, J., and B. Oliver, 2011Microarrays, deep sequencing and the true
measure of the transcriptome. BMC Biol. 9: 34.
McIntyre, L., L. Bono, A. Genissel, R. Westerman, D. Junk et al., 2006
specific expression of alternative transcripts in Drosophila. Genome Biol.
McIntyre, L. M., and K. K. Lopiano, A. M. Morse, V. Amin, A. L. Oberg et al.,
2011 RNA-seq: technical variability and sampling. BMC Genomics 12: 293.
McManus, C. J., J. D. Coolon, M. O. Duff, J. Eipper-Mains, B. R. Graveley
et al., 2010 Regulatory divergence in Drosophila revealed by mRNA-
seq. Genome Res. 20: 816–825.
Meyer, K. B., A.-T. Maia, M. O'Reilly, A. E. Teschendorff, S.-F. Chin et al.,
2008 Allele-specific up-regulation of FGFR2 increases susceptibility to
breast cancer. PLoS Biol. 6: e108.
Neter, J., W. Wasserman, and M. H. Kutner, 1990
Models: Regression, Analysis of Variance, and Experimental Designs. Ir-
win, Homewood, Ill.
Nica, A. C., and E. T. Dermitzakis, 2008
the genetic basis of complex disorders. Hum. Mol. Genet. 17: R129–R134.
Nica, A. C., S. B. Montgomery, A. S. Dimas, B. E. Stranger, C. Beazley et al.,
2010Candidate causal regulatory effects by integration of expression
QTLs with complex trait genetic associations. PLoS Genet. 6: e1000895.
Parisi, M., R. Nuttall, D. Naiman, G. Bouffard, J. Malley et al., 2003
of genes on the Drosophila X chromosome showing male-biased
expression. Science 299: 697–700.
Pastinen, T., R. Sladek, S. Gurd, A. A. Sammak, B. Ge et al., 2004
of genetic and epigenetic variation affecting human gene expression.
Physiol. Genomics 16: 184–193.
Pickrell, J. K., J. C. Marioni, A. A. Pai, J. F. Degner, B. E. Engelhardt et al.,
2010 Understanding mechanisms underlying human gene expression
variation with RNA sequencing. Nature 464: 768–772.
Rabbee, N., and T. P. Speed, 2006A genotype calling algorithm for
Affymetrix SNP arrays. Bioinformatics 22: 7–12.
Ranz, J. M., C. I. Castillo-Davis, C. D. Meiklejohn, and D. L. Hartl,
2003 Sex-dependent gene expression and evolution of the Drosophila
transcriptome. Science 300: 1742–1745.
Rifkin, S. A., J. Kim, and K. P. White, 2003
the Drosophila melanogaster subgroup. Nat. Genet. 33: 138–144.
Rockman, M. V., and L. Kruglyak, 2006
Nat. Rev. Genet. 7: 862–872.
Ross, D. T., U. Scherf, M. B. Eisen, C. M. Perou, C. Rees et al.,
2000Systematic variation in gene expression patterns in human cancer
cell lines. Nat. Genet. 24: 227–235.
Schena, M., D. Shalon, R. W. Davis, and P. O. Brown, 1995
monitoring of gene expression patterns with a complementary DNA
microarray. Science 270: 467–470.
Applied Linear Statistical
Using gene expression to investigate
Evolution of gene expression in
Genetics of global gene expression.
Serre, D., S. Gurd, B. Ge, R. Sladek, D. Sinnett et al., 2008
expression in the human genome: a robust approach to identify genetic
and epigenetic cis-acting mechanisms regulating gene expression. PLoS
Genet. 4: e1000006.
Silverman, B. W., 1986Density Estimation for Statistics and Data Analysis.
Champman and Hall, London, UK.
Stamatoyannopoulos, J. A., 2004 The genomics of gene expression.
Genomics 84: 449–457.
Stranger, B. E., A. C. Nica, M. S. Forrest, A. Dimas, C. P. Bird et al.,
2007Population genomics of human gene expression. Nat. Genet.
Szabó, P. E., and J. R. Mann, 1995Biallelic expression of imprinted genes in
the mouse germ line: implications for erasure, establishment, and
mechanisms of genomic imprinting. Genes Dev. 9: 1857–1868.
Telonis-Scott, M., A. Kopp, M. L. Wayne, S. V. Nuzhdin, and L. M. McIntyre,
2009 Sex-specific splicing in Drosophila: widespread occurrence,
tissue-specificity, and evolutionary conservation. Genetics 181:
Thompson, J. D., D. G. Higgins, and T. J. Gibson, 1994
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res. 22: 4673–4680.
Verhoeven, K. J. F., K. L. Simonsen, and L. M. McIntyre, 2005
false discovery rate control: increasing your power. Oikos 108: 643–647.
Verlaan, D. J., S. Berlivet, G. M. Hunninghake, A.-M. Madore, M. Larivière
et al., 2009 Allele-specific chromatin remodeling in the ZPBP2/
GSDMB/ORMDL3 locus associated with the risk of asthma and auto-
immune disease. Am. J. Hum. Genet. 85: 377–393.
Wang, D., H. Chen, K. M. Momary, L. H. Cavallari, J. A. Johnson et al.,
2008 Regulatory polymorphism in vitamin K epoxide reductase com-
plex subunit 1 (VKORC1) affects gene expression and warfarin dose
requirement. Blood 112: 1013–1021.
Wayne, M. L., M. Telonis-Scott, L. M. Bono, L. Harshman, A. Kopp et al.,
2007 Simpler mode of inheritance of transcriptional variation in
male Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 104: 18577–
Wittkopp, P., B. Haerum, and A. Clark, 2008
lying expression differences within and between Drosophila species. Nat.
Genet. 40: 346–350.
Wittkopp, P. J., B. K. Haerum, and A. G. Clark, 2004
in cis and trans gene regulation. Nature 430: 85–88.
Wolfner, M. F., 1997 Tokens of love: functions and regulation of
drosophila male accessory gland products. Insect Biochem. Mol. Biol.
Zhang, K., J. B. Li, Y. Gao, D. Egli, B. Xie et al., 2009
typing reveals tissue-specific and allele-specific gene expression in human.
Nat. Methods 6: 613–618.
Zhang, X., and J. O. Borevitz, 2009Global analysis of allele-specific ex-
pression in Arabidopsis thaliana. Genetics 182: 943–954.
Regulatory changes under-
Digital RNA allelo-
Communicating editor: R. Kulathinal
| Y. Yang et al.