Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing.
ABSTRACT One of the key advantages of using Drosophila melanogaster as a genetic model organism is the ability to conduct saturation mutagenesis screens to identify genes and pathways underlying a given phenotype. Despite the large number of genetic tools developed to facilitate downstream cloning of mutations obtained from such screens, the current procedure remains labor intensive, time consuming, and costly. To address this issue, we designed an efficient strategy for rapid identification of heterozygous mutations in the fly genome by combining rough genetic mapping, targeted DNA capture, and second generation sequencing technology. We first tested this method on heterozygous flies carrying either a previously characterized dac(5) or sens(E2) mutation. Targeted amplification of genomic regions near these two loci was used to enrich DNA for sequencing, and both point mutations were successfully identified. When this method was applied to uncharacterized twr mutant flies, the underlying mutation was identified as a single-base mutation in the gene Spase18-21. This targeted-genome-sequencing method reduces time and effort required for mutation cloning by up to 80% compared with the current approach and lowers the cost to <$1000 for each mutant. Introduction of this and other sequencing-based methods for mutation cloning will enable broader usage of forward genetics screens and have significant impacts in the field of model organisms such as Drosophila.
- SourceAvailable from: Michael Anthony Gonzalez[Show abstract] [Hide abstract]
ABSTRACT: Forward genetic screens in Drosophila melanogaster using ethyl methanesulfonate (EMS) mutagenesis are a powerful approach for identifying genes that modulate specific biological processes in an in vivo setting. The mapping of genes that contain randomly-induced point mutations has become more efficient in Drosophila thanks to the maturation and availability of many types of genetic tools. However, classic approaches to gene mapping are relatively slow and ultimately require extensive Sanger sequencing of candidate chromosomal loci. With the advent of new high-throughput sequencing techniques, it is increasingly efficient to directly re-sequence the whole genome of model organisms. This approach, in combination with traditional chromosomal mapping, has the potential to greatly simplify and accelerate mutation identification in mutants generated in EMS screens. Here we show that next-generation sequencing (NGS) is an accurate and efficient tool for high-throughput sequencing and mutation discovery in Drosophila melanogaster. As a test case, mutant strains of Drosophila that exhibited long-term survival of severed peripheral axons were identified in a forward EMS mutagenesis. All mutants were recessive and fell into a single lethal complementation group, which suggested that a single gene was responsible for the protective axon degenerative phenotype. Whole genome sequencing of these genomes identified the underlying gene ect4. To improve the process of genome wide mutation identification, we developed Genomes Management Application (GEM.app, https://genomics.med.miami.edu), a graphical online user interface to a custom query framework. Using a custom GEM.app query, we were able to identify that each mutant carried a unique non-sense mutation in the gene ect4 (dSarm), which was recently shown by Osterloh et al. to be essential for the activation of axonal degeneration. Our results demonstrate the current advantages and limitations of NGS in Drosophila and we introduce GEM.app as a simple yet powerful genomics analysis tool for the Drosophila community. At a current cost of <$1,000 per genome, NGS should thus become a standard gene discovery tool in EMS induced genetic forward screens.Biology 12/2012; 1(3):766-77.
- [Show abstract] [Hide abstract]
ABSTRACT: Natural variants of crops are generated from wild progenitor plants under both natural and human selection. Diverse crops that are able to adapt to various environmental conditions are valuable resources for crop improvements to meet the food demands of the increasing human population. With the completion of reference genome sequences, the advent of high-throughput sequencing technology now enables rapid and accurate resequencing of a large number of crop genomes to detect the genetic basis of phenotypic variations in crops. Comprehensive maps of genome variations facilitate genome-wide association studies of complex traits and functional investigations of evolutionary changes in crops. These advances will greatly accelerate studies on crop designs via genomics-assisted breeding. Here, we first discuss crop genome studies and describe the development of sequencingbased genotyping and genome-wide association studies in crops. We then review sequencing-based crop domestication studies and offer a perspective on genomics-driven crop designs. Expected final online publication date for the Annual Review of Plant Biology Volume 65 is April 29, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.Annual Review of Plant Biology 11/2013; · 18.71 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of Next-Generation Sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of 1.) a sequenced genome; 2.) a strongly penetrant and easily measurable phenotype; and 3.) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the post genomic era.Genetics 07/2013; · 4.87 Impact Factor
Rapid identification of heterozygous mutations
in Drosophila melanogaster using genomic
Hui Wang,1,2Abanti Chattopadhyay,2Zhe Li,3Bryce Daines,2Yumei Li,1,2Chunxu Gao,2
Richard Gibbs,1,2Kun Zhang,3and Rui Chen1,2,4,5
1Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030, USA;2Department of Molecular and Human
Genetics, Baylor College of Medicine, Houston, Texas 77030, USA;3Department of Bioengineering, University of California at San
Diego, La Jolla, California 92093, USA;4Program in Developmental Biology, Baylor College of Medicine, Houston,
Texas 77030, USA
One of the key advantages of using Drosophila melanogaster as a genetic model organism is the ability to conduct saturation
mutagenesis screens to identify genes and pathways underlying a given phenotype. Despite the large number of genetic
tools developed to facilitate downstream cloning of mutations obtained from such screens, the current procedure remains
labor intensive, time consuming, and costly. To address this issue, we designed an efficient strategy for rapid identification
of heterozygous mutations in the fly genome by combining rough genetic mapping, targeted DNA capture, and second
generation sequencing technology. We first tested this method on heterozygous flies carrying either a previously char-
sequencing, and both point mutations were successfully identified. When this method was applied to uncharacterized twr
sequencing method reduces time and effort required for mutation cloning by up to 80% compared with the current
approach and lowers the cost to <$1000 for each mutant. Introduction of this and other sequencing-based methods for
mutation cloning will enable broader usage of forward genetics screens and have significant impacts in the field of model
organisms such as Drosophila.
[Supplemental material is available online at http:/ /www.genome.org. The sequence data from this study have been
submitted to the NCBI Sequence Read Archive (http:/ /www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession no.
The availability of genetic tools in Drosophila to generate, screen,
and characterize mutations with phenotypes of interest makes it
one of the most powerful genetic model organisms. One of the
most commonly used mutagens, ethyl methane sulfonate (EMS),
has been widely used in genetic screens, and mutations identified
in more than 3000 genes have been recorded in FlyBase (http://
flybase.org/). In order to clone an EMS or otherchemical mutagen-
induced novel mutation, it is often necessary to reduce the can-
fine genetic mapping (H Bellen, pers. comm.).However, due to the
large number of flies that must be scored in order to achieve such
resolution, the fine mapping step is quite labor intensive and
costly. Additionally, for some genomic regions, it is not always
possible to map a mutation to a small interval due to limitations
such as low recombination rates and lack of mapping stocks.
Several molecular methods have been proposed for cloning
EMS mutations without the need for genetic mapping. One
DNA cleavage and can be used to identify changes in genes of in-
It is ideal for isolating alleles of given genes from a large collection
of EMS-induced mutants; however, this method is not well suited
for identifying mutations obtained from forward genetic screens,
since candidate genes need to be predefined for testing using
TILLING. Another approach of mutation cloning is to take ad-
vantage of the recent development of second-generation sequenc-
ing technology. Rapid progress in sequencing technologies has
dramatically increased the throughput and reduced the cost of
DNA sequencing (Mardis 2008a,b; Shendure and Ji 2008; Ansorge
2009). Recently, identificationof homozygous mutationsby direct
whole-genome sequencing has been reported (Sarin et al. 2008;
Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009).
Mutations in genes that are important for development, however,
are often homozygous lethal, and it is necessary to detect hetero-
zygous mutations. The detection of heterozygous mutations re-
quires much more sequencing coverage (Bentley et al. 2008; Ley
et al. 2008; Wheeler et al. 2008). We estimate that about 303 se-
quencing coverage is necessary to detect heterozygous mutations
with high sensitivity (>95%) and accuracy (error rate <10?6)
(Supplemental Fig. 1). At the current cost of about $1500 for every
103 sequencing coverage of the Drosophila genome, the direct
whole-genome sequencing approach is still too expensive for
and multiple alleles often need to be sequenced in parallel to dis-
tinguish single nucleotide polymorphisms (SNPs) and other
changes from causative mutations.
When a cloning by sequencing approach is used, rough
genetic mapping of the mutation is often highly valuable, as
it can greatly reduce downstream data analysis efforts. Since
Article published online before print. Article and publication date are at
20:981–988 ? 2010 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/10; www.genome.org
approximately one base mutation is induced per 400 kb when flies
are treated with 25 mM EMS, a commonly used experimental
condition, a total of about 400 base changes exist in each mutant
fly (Cooper et al. 2008; Blumenstiel et al. 2009). Mapping of the
mutationcan greatlyreducethe number of candidatechangesthat
need to be analyzed. Although fine genetic mapping is quite labor
intensive, intermediate levels of genetic mapping where a muta-
tion is mapped to within a few megabases (Mb) can be achieved
efficiently using methods such as P element or SNP mapping in
Drosophila (Zhai et al. 2003; Chen et al. 2008). Once a mutation is
mapped to an interval of several megabases or less, it is then nec-
essary to sequence only the candidate region (often <1% of the
genome) instead of the entire genome, resulting in significant cost
reduction. Recently, several DNA enrichment methods have been
developed that enable enrichment of DNA from targeted regions
2009; Ng et al. 2009; Okou et al. 2009). With optimized probe de-
sign and capture conditions, the vast majority of a targeted region
(>95%) can be efficiently enriched (H Wang and R Chen, unpubl.).
Among the numerous methods for targeted DNA enrichment,
padlock capture, which relies on a combination of oligonucleotide
hybridization and enzymatic activity, shows the greatest specific-
probes that contain target-specific capturing arms can be annealed
to the target regions. DNA synthesis using the target regions as
templates will generate circular DNA, which is selectively ampli-
fied by PCR using commonsequences flanking the capturearms as
primers. A key feature of padlock probes is that they can be
regenerated by PCR, thereby greatly reducing costs when multiple
enrichments are needed, as in the case of sequencing com-
plementing mutations of the same locus.
To test the feasibility of identifying heterozygous mutations
by targeted genome sequencing, we first applied the proposed
method to two known mutations, dac5and sensE2. Exons within
0.5 Mb flanking the two known mutations were enriched and se-
GAII systems. We found that the causative point mutations could
be reliably identified by either platform using only a small fraction
of each platform’s capacity. Next, we applied this method to un-
cover the mutation in a previously uncharacterized mutant, twr1,
and identified a missense mutation in the Spase18-21 gene. Two
lines of evidence support that the identified mutation in Spase18-
identified mutations in Spase18-21 in two additional twr mutant
alleles, twr2and twr11. Second, a P-element insertion that fails to
complement the twr1mutant allele maps to the 59 end of the
Spase18-21 gene. In summary, we conclude that combining rough
genetic mapping, DNA targeted capture, and second generation
sequencing is highly cost effective and immediately applicable to
the forward genetic screen.
We toidentifya strategy forcloning ofthese lethalmutationsinthe
Drosophila genome. As an alternate approach to whole-genome se-
quencing, targeted genomic sequencing offers the potential of sig-
nificant cost reduction by reducing the portion of the genome se-
quenced (Li et al. 2009b). This is particularly suitable for model
organisms in which genetic mapping can rapidly reduce candidate
loci to a reasonable genomic interval. In Drosophila, the combina-
tion of genetics markers, P-element insertions, and chromosomal
deficiency and duplication stocks, make it possible to map a lethal
mutation within a few hundred kilobase interval efficiently. As
be sequenced to identify an underlying mutation. Therefore, sig-
nificant cost reduction can be potentially achieved if the small in-
terval can be enriched from the genome and sequenced.
Capture sequencing as a cost-effective strategy for detecting
To test this approach, we performed targeted sequencing on two
previously characterized mutant alleles, one in the gene senseless
(sens), sensE2, and the other in dachshund (dac), dac5(Fig. 1B,C).
Using the padlock method, DNA oligo probes covering all exons
within the 0.5-Mb region centered on each of these two mutations
were designed and used to enrich DNA from adult heterozygous
mutant flies (Fig. 1A). As shown in Figure 1B, sensE2is a nonsense
mutation caused by a G to A transition in the sens gene at
13,393,009 bp on chromosome 3L (version5.1). Flies homozygous
for the sensE2mutation are embryonic lethal. To enrich DNA for
the annotatedexons in the 0.5-Mb region surrounding sens, a total
of 257 amplicons covering 86,435 exon bases were designed
(Supplemental File 1). Using this probe set, DNA from sensE2het-
erozygous flies was enriched and about 37,000 sequencing reads
the targeted region, giving an average sequencing coverage of 443
with median coverage of 383. At least 13 coverage was observed
coverage. In total, 62 SNPs were detected, including the known
SNP. As shown in Figure 1B, a total of 33 reads covering the mu-
tated base in sensE2were obtained, with 21 reads representing the
wild-type allele and 12 representing the mutant allele, resulting in
a SNP with a quality score of 84. This finding was confirmed by
direct Sangersequencing of a PCRproductcontainingthemutated
base (Fig. 1B, arrow). Similarly, for the 0.5-Mb genomic locus
around dac, a total of 249 amplicons covering 112,284 annotated
exon bases were designed. Capture sequencing was performed on
dac5heterozygous DNA, which contains a G-to-A transition at
16,472,530 bp on chromosome 2L (version 5.1). A total of 24,530
454 sequencing reads were obtained and mapped to the targeted
region, which was equivalent to an average coverage of 223 with
median coverage of 113. A total of 97,917 (87%) bases were cov-
ered at least once while 64,580 (57.5%) of all targeted bases had
103 coverage or higher and 11 SNPs were detected in total. A total
of 10 reads covering the known dac5mutant allele were obtained,
with four reads representing the wild-type allele and six reads
representing the mutant allele, resulting in a SNP with a quality
score of 87 (Fig. 1C). Consistent with this result, the heterozygous
mutation was further confirmed by directly sequencing the PCR
product (Fig. 1C, arrow). Taken together, these results demonstrate
that previously known mutant alleles in heterozygous flies can be
identified by capture sequencing.
sensE2mutant flies, although the mean coverage is 443, ;25% of
targeted bases have a coverage of 103 or lower, and 7.4% of all
targeted bases are not sequenced at all. Therefore, we asked
whether higher sequencing coverage would increase the portion
of bases with sufficient sequencing coverage. DNA from two
independent capture experiments on the sensE2heterozygous flies
was sequenced on the Illumina GAII platform at 903 and 1403
coverage. As expected, an increase in sequencing depth increases
the percentage of bases with 103 coverage (Supplemental Fig. 1C).
Wang et al.
generation sequencing technologies. Padlock capture technology requires probes that contain two target-specific capturing arms (green) connected by
a common linker (red). The unique targeting arms of individual targeting oligonucleotides are designed to hybridize immediately upstream and
downstream from each exon (purple bars) of interest. Hybridization to genomic DNA is followed by an enzymatic gap filling and ligation step, such that
a copy of the sequence of interest is incorporated into a circle (purple dashed line). Thenthe enriched DNA is PCR amplified and is used to prepare libraries
for the next-generation platform sequencing. (B) sensE2and (C) dac5mutation detection. Reads alignment at the mutation is shown between the vertical
with the heterozygous mutated base indicated by the arrow.
Two known mutations were detected using padlock capture sequencing. (A) Flowchart of mutation detection by padlock capture and next-
Mutation identification by genome targeted sequencing
least 103 coverage, while only 3.9% of the bases remain un-
covered. In addition, the coverage profile is very similar between
these two capture experiments (data not shown), suggesting that
the method is quite robust and reproducible. As the capture region
of sensE2represents just 0.05% of the genome, the total amount of
sequence data generated at 1403 coverage of this region is equiv-
alent to only ;0.073 coverage of the whole genome.
Identification of novel mutations using the capture
To determine whether the capture sequencing method is applica-
twr mutant flies. twr mutants were first identified through an EMS
mutagenesis screen of the ANTP-C region (Lewis et al. 1980;
Hazelrigg and Kaufman 1983). In this screen, multiple twr alleles
were recovered with three stocks that are available from the
Bloomington stock center including twr1, twr2, and twr11(Fig. 2A).
Although twr mutant flies are homozygous lethal, trans heterozy-
gous twr mutant flies exhibit disorganization of ommatidia with
degenerative photoreceptors (Fig. 2C,E). The twr mutant has been
mapped to cytological position 84A, but the molecular nature of
these mutations remains unknown. To identify the underlying
mutations in twr, we designed a capture probe set spanning the
84A region, starting from 2.27 to 2.73 Mb on chromosome 3R
(Supplemental File 1). This probe set amplifies a total of 285
amplicons containing74,001 bp of exon sequences in the targeted
region. DNA enriched by this probe set was sequenced using the
GAII platform and a total of 206,729 32-bp reads were generated
and mapped to the targeted region, resulting in an average of 743
coverage of the targeted region. As a result, only 4.8% of the tar-
geted region was missing, while 78% of the targeted region had
>103 sequence coverage. Interestingly, a large number of changes
were identified, including 105 heterozygous changes and 21 ho-
mozygous changes, most of which probably reflect the prevalence
of SNPs in the Drosophila genome. A large portion of these po-
tential variants were synonymous changes, while only 26 resulted
in amino acid changes (Supplemental File 2). To distinguish po-
tential mutations from SNPs, it would be best to obtain sequences
from the parental strain. Unfortunately, the twr1allele was gener-
ated more than 20 yr ago, and the parental strain used for muta-
genesis is no longer available. To solve this problem, capture se-
quencing was performed on heterozygous flies carrying an
independently derived twr mutant allele, twr2. As both twr1and
twr2alleles were induced from the same parental strain, these two
mutant fly strains should share parental SNPs. Indeed, among the
26 heterozygous variants identified in the heterozygous twr1flies,
24 are also identified in the twr2flies, indicating that thesevariants
are likely to be SNPs inherited from their parental strain (Supple-
mental File 2). Only two single-base variants appear to be twr1
specific and were likely induced during mutagenesis, one at base
position 2,485,103 A ! Tand the second one at 2,518,875 G ! A.
Variant at base position 2,518,875 results in amino acid change
from Gly to Ser in gene Ccp84Ad (chitin cuticular protein at 84Ad).
Another mutation at base 2,485,103 results in a significant change
in gene Spase18-21. Among the 221 reads covering this position,
135 reads contains the wild-type base (A) and the remaining 86
were mutated to base T (Fig. 2F). As a result, the normal stop codon
of the Twr protein (Fig. 2F). Interestingly, the amino acid sequence
of the last coding exon and the position of the stop codon of
Spase18-21 are completely conserved in 12 Drosophila species (Fig.
2G). Further support that the variant in Spase18-21 is the causative
mutationforthetwr phenotype comesfromsequencingof thetwo
disrupts the splicing acceptor site of exon 3 of the Spase18-21 gene
was identified in twr2mutant flies (Fig. 2A; data not shown). These
data suggested that twr flies indeed carry mutations in the Spase18-
21 gene. This conclusion is further supported by characterization
of a homozygous lethal P-element insertion, twr05614(Fig. 2A).
twr05614is an allele of twr that fails to complement twr1mutant.
Inverse PCR confirms the insertion site at position 3R: 2,483,680
bp, just 76 bp 59 of the Spase18-21 gene as recorded in FlyBase (Fig.
2A; data not shown). Furthermore, when the P-element is mobi-
lized, wild-type reverted flies are recovered, indicating that the
phenotype observed in twr1/twr05614is caused by the P-element
insertion. Taken together, we conclude that twr mutant flies carry
mutations in Spase18-21.
Forward genetic approaches are widely used from bacteria to mice
and play an essential role in modern biology by identifying genes
with interesting phenotypes. In model organisms, the forward
genetic screen remains one of the most powerful tools to study
the cloning step. It can take months of effort to identify the mo-
tools that are available in Drosophila. In our study, we demonstrate
next-generation sequencing is a viable method for rapid and cost-
sequencing to generate billions of bases at low cost makes it effi-
cient to sequence a megabase region.As a result, only intermediate
levels of genetic mapping are needed to narrow down a muta-
tion to within a few megabases, thereby eliminating the labor-
intensive, time-consuming genetic fine-mapping step. As a result,
the time for cloning a mutation can be shortened dramatically
from 6 mo to ;2 mo along with significantly less effort. Second,
the DNA capture method makes it possible to enrich specific ge-
nomic regions for sequencing. To achieve high sensitivity (>95%)
and specificity, we have estimated that 303 sequencing coverage
is needed to identify heterozygous mutations with high accuracy
(less than one error per megabase) and sensitivity (>95%) for 90%
of the genome (Supplemental Fig. 1). Furthermore, to increase
the likelihood of identifying changes and distinguishing pre-
existing SNPs from true mutations, it is best to sequence multiple
alleles within the same complementation group, along with their
respective controls. Therefore, the choice of sequencing approach
is primarily driven by the sequencing cost. Even at the currently
low sequencing cost of $750 per gigabase, detection of mutations
by direct whole-genome sequencing is quite costly. Together
with sequencing library construction cost, $4500 per stain at 303
sequence coverage, a total of $14,400 is needed to perform whole-
genome sequencing for two alleles with their parental strain as
control (Table 1). In contrast, the DNA capture technology used
in our study offers greatly reduced sequencing costs while in-
a 0.5-Mb region surrounding two known mutations, dac5and
sensE2, were captured and sequenced. Even with high coverage of
1403 forthe targetedregion,the totalamountof sequenceneeded
Wang et al.
is still <1% that of whole-genome sequencing. As a result, the
current sequencing cost for captured DNA is <$400 per strain,
bringing the total cost below $2000 for characterizing two alleles
plus the parental strain (Table 1). Furthermore, even with a 10-fold
reduction of the current sequencing cost, the capture sequencing
approach will still offer significant savings over the whole-genome
shotgun approach (Table 1, projected cost columns). Third, the
high coverage obtained from capture sequencing ensures sensi-
tivity and accuracy in detecting mutations. At 403 sequencing
coverage, 57.5% and 75.2% of bases enriched in dac5and sensE2
sequencing showed >103 coverage, respectively. The portion of
highly covered bases can be further increased by simply perform-
sens locus were sequenced at least 10 times when the sequence
coverage was increased to 1403. This high coverage is essential for
the accurate identification of mutations.
detected within the Spase18-21 gene in three different twr alleles, twr1, twr2, and twr11, using capture sequencing. Alleles twr1and twr11shared the same
mutation of an A-to-T transition at position 3R:2,485,014. twr2was found to have a different transition of G to A at position 3R: 2,484,724. A P-element
twr that failed to complement twr1was found by inverse PCR to be located 76 bp downstream from the Spase18-21 start site at position
3R:2,483,680. Compared with the wild type (B,D), twr1/twr2transheterozygous adult flies (C,E ) show rough eye phenotype and missing photoreceptors.
(F ) Alignment of reads at the position of the twr1mutation is shown. Alignment of capture sequencing reads covering the heterozygous mutation in the
twr1is shown on the left. The mutated base pair is highlighted in red. Direct Sanger sequencing was performed to confirm the mutation in twr1, with the
heterozygousmutatedbaseindicated bythearrow.Theadditionof12aminoacidsthatwere causedbythemutation arealsoshown.(G)Alignment ofthe
SPASE18-21 amino acid sequence within several Drosophila species is shown, indicating a high degree of identity.
Padlock capture was successfully used to identify novel mutations. (A) The genomic locus of twr and its alleles are shown. Mutations were
Mutation identification by genome targeted sequencing
Regardless of the sequencingstrategy, roughgenetic mapping
data will be highly desired during data analysis and should be in-
cluded as a key part of mutation cloning. There is approximately
one SNP per 200 bp in each Drosophila strain (Platts et al. 2009).
Indeed, a large number of SNPs have been observed when mutant
fly sequences are compared with that of the reference genome. For
example, in the twr1region we found 126 nucleotide changes, of
which 26 caused changes in the amino acid sequence. Therefore,
to distinguish SNPs from mutations, it is crucial to have the pa-
rental strain sequenced as a control. A polymorphism should exist
in both the parent and the mutant, while EMS-induced mutations
are mutant specific. To minimize complications such as sponta-
neous mutations accumulating in the parental stocks, it is rec-
of mutagenesis for reference. In cases where the parental strain is
no longer available, parallel sequencing of multiple alleles can also
serve this purpose. Another issue during data analysis is the fact
that several hundred base changes can be induced by EMS across
the genome. Several measures can be implemented to facilitate
identification of the causative mutation. First, a large number of
EMS-induced changes can be excluded by genetic mapping data.
Only changeswithin the targeted region are relevant. Second, data
obtained from multiple independent alleles can be compared to
identify shared mutant genes. As EMS-induced mutations are
random, this will greatly reduce false positives. Third, a large
number of changes will be benign. A base change should be ex-
cluded if it fails to affect an amino acid or an mRNA splice site. In
addition, as more Drosophila sequencing is carried out, the estab-
lishment of a database cataloging common SNPs will be a critical
step to facilitate downstream analysis.
Through capture sequencing, we were able to identify muta-
tions in both dac5and sensE2heterozygous flies. In addition, we
have identified that the novel underlying mutation in twr1lies
within the Spase18-21 gene. The molecular nature of the twr1
mutation is a base mutation at the stop codon, resulting in an
addition of 12 amino acids to the C terminus of the protein. It is
interesting that such a small change in the SPASE18-21 protein in
twr1has such a dramatic impact on its normal function. SPASE18-
21 is a subunit of a signal peptidase that catalyzes the cleavage of
signal peptides within the endoplasmic reticulum. The amino acid
sequence of SPASE18-21 is nearly identical across all 12 sequenced
Drosophila species. Therefore, it is conceivable that small changes
in the protein might lead to an alteration in the protein’s tertiary
multisubunit signal peptidase complex, thereby dramatically re-
ducing the enzymatic activity of this complex.
We found that typically 10%–15% of the bases are not well
covered by capture sequencing using padlock probes. The effi-
ciency can probably be improved by several methods. First, the
probe design, adding blocking oligos at overrepresented regions,
and adding additional capture probes for underrepresented re-
gions, has been shown to improve capture results (Li et al. 2009b).
Second, a set of padlock probes specific for under-represented re-
gions can be designed and used in a separate experiment in addi-
tion to the original kit. Without competition from overenriched
regions, under-represented regions are likely to be captured more
efficiently. Third, other enrichment methods such as DNA hy-
bridization liquid capture can be used either in conjunction with
the padlock approach or independently. Near complete capture
can be achieved with DNA hybridization methods when the probe
design is optimized (H Wang and R Chen, unpubl.). Finally, to
improve the sensitivity of detecting mutations, capture sequenc-
ing can be conducted for all alleles of the same complementation
group when available. This is feasible, as the cost of capture se-
quencing is quite low. As independent alleles usually harbor mu-
tations at different positions in a given gene, the problem of
missing small portions of a gene sequence is minimized.
In summary, with the development of capture probe sets
across the entire genome, establishment of a SNP database, and
further development of sequencing technologies and data analysis
tools, mutation cloning in Drosophila will become straightforward
and cost efficient. Compared with the current genetic fine-mapping
plus Sanger sequencing approach, which typically requires at least
6 mo and costs at least $1500 in reagent alone, our proposed
methodcanbe completedwithin2 mowithpartialeffortandcosts
in Drosophila research will be further broadened, including the
generation of a complete collection of EMS-induced mutations.
Genetic mapping can be conducted in most model organisms ef-
ficiently, and, hence, this approach should also be readily appli-
cable to other model organisms such as mice.
Fly genetics and DNA preparation
All flies used in this study were maintained on standard Drosophila
medium in a 25°C room with a light cycle of 12 h light and 12 h
dark. Genomic DNA from flies was prepared using Buffer A (100
mM Tris-HCl at pH 7.5, 100 mM EDTA, 100 mM NaCl, 0.5% SDS),
followed by LiCl/KAc incubation and ethanol precipitation.
Padlock probe design
Targets were defined as protein-coding sequences of three candi-
date regions in the Drosophila genome (US National Center for
Cost comparison between whole-genome shotgun at 303 and capture sequencing
Cost per strain
Total cost (three strains)
$0 $0 $200
Calculation is conducted based on $750 per gigabase sequencing in January 2010 and projected cost between $70 and $110 per gigabase in January
2011. Furthermore, with the DNA capture approach, the sequencing library construction step will be reduced to a single PCR amplification step, resulting
in significant savings in both cost and time. Overall, even with the projected >10-fold reduction of sequencing cost, the savings offered by capture is still
Wang et al.
986 Genome Research
Biotechnology Information [NCBI], April 2006). We developed
a probe design algorithm to search for an optimal set of padlock
probes covering an arbitrary set of nonrepetitive genomic targets
(Porreca et al. 2007). This algorithm weights candidate probes
based on several sequence features that were previously not con-
sidered in eMIP probe design, including the melting temperature,
size of the capturing arms, and gap sizes (AJ Gore and K Zhang,
unpubl., in prep.). The average size of the target regions is 140 bp,
with a standard deviation of 14.6. Sequences for the designed
probes can be found in Supplemental File 1. These Agilent oligos
were released and converted to padlock probes using the protocol
described previously (Deng et al. 2009).
Capture of targeted sequences
We hybridized targeting oligos to genomic DNA in 20 mL of 13
Ampligase buffer (Epicentre) with 200 ng of genomic DNA and
2 ng of targeting oligos, incubating the reactions at 95°C for
2 min and 60°C for 20 h. Then, we added 1 mL of gap-filling mix
and 0.5 U of Ampligase in 13 Ampligase buffer), and incubated
the reaction at 60°C for 20 h. To degrade linear species, we added
2 mL of exonuclease mix (containing 50 U of exonuclease I and
500 U of exonuclease III; New England BioLabs), and incubated
the reaction at 37°C for 2 h and then at 95°C for 2 min.
Amplification of enriched DNA
Enriched DNA was amplified by PCR reaction. A total of 1 mL of
captured DNA was used as template in a 12.5-mL reaction. PCR
conditions were: 95°C for 15min, 30 cycles of 95°C for 30 sec, 55°C
for 30 sec, 72°C for 30 sec, and, finally, 72°C for 5 min. PCR prod-
ucts were separated on a 2% agarose gel. We recovered amplicons
corresponding to the expected size range (170–210 bp), purified
them, and resuspended the products in 20 mL of TE (pH 8.0).
The purified PCR amplicons were directly used as the DNA tem-
plate for the 454 Life Sciences (Roche) library. The 454 GS FLX
Titanium libraries were prepared according to the manufacturer’s
protocol. To prepare Illumina libraries, purified PCR amplicons
were first digested with MmeI: 16 units of MmeI (2 U/mL; New
4 at 37°C for 1 h. Digestions were again column purified and
digested with 3 U of USER enzyme (1 U/mL; New England BioLabs)
in 13 S1 nuclease buffer at 37°C for 10 min. The fragmented DNA
was column purified and used as DNA template for the Illumina
library. Illumina librarieswere generated by followingthe Illumina
pair-end library preparation protocol.
with SAMtools SNP calling for a custom capture SNP-calling
html; Kent 2002; Li et al. 2009a). 454 Life Sciences (Roche) and
Illumina GAII sequencing reads were anchored to the Drosophila
genome (dm3) using BLAT parameters appropriate to each plat-
form’s read length. Reads with a unique or ‘‘best’’ hit were then
combined and converted to SAM/BAM format. SAMtools was used
to generate pileup files with the ‘‘pileup –cf’’ option and the sam-
tools.pl script was used to identify candidate SNPs with these
paramaeters: ‘‘varFilter –D 500.’’ Increasing the maximum read
depth is necessary due to the enrichment of captured regions. As
an example, parameters for Illumina GAII BLAT mapping were:
followed by cross_match alignment with flags: ‘‘?minscore = 24
–bandwidth = 6 –gap_init = ?2 –penalty = ?1 –gap_ext = ?1 –raw
–masklevel = 0.’’
To confirm mutations identified by the 454 Life Sciences (Roche)
and Illumina GAII parallel sequencing, a direct PCR sequencing
approach was used. Specific PCR primers were designed sur-
rounding the SNPs, and target SNPs were amplified. PCR products
were purified with ExoSAP-IT (USB Corp.). Sequencing was per-
formed using an ABI PRISM Big Dye Terminator Cycle Sequencing
Ready Reaction Kit v3.1 according to the manufacturer’s recom-
mendations. The ABI 3700 capillary electrophoresis system was
used to carry out the electrophoretic separations, and sequencer
software was used to analyze the data.
We thank Graeme Mardon for providing dac5and sensE2flies, for
scientific discussion, and for review of the manuscript. We thank
thank Hugo Bellen for critical reading of the manuscript. Finally,
we thank the staff of the Human Genome Sequencing Center who
performed the sequencing of genomic libraries. B.D. is supported
by training grant T32 EYO7102-16. H.W. is supported by postdoc
fellowship EY19430-01. This work is supported by the Retinal Re-
search Foundation and NEI/NIH grant R01EY016853 to R.C.
TA, Middle CM, Rodesch MJ, Packard CJ, et al. 2007. Direct selection of
human genomic loci by microarray hybridization. Nat Methods 4: 903–
Ansorge WJ. 2009. Next-generation DNA sequencing techniques. New
Biotechnol 25: 195–203.
Bau S, Schracke N, Kranzle M, Wu H, Stahler PF, Hoheisel JD, Beier M,
Summerer D. 2009. Targeted next-generation sequencing by specific
capture of multiple genomic loci using low-volume microfluidic DNA
arrays. Anal Bioanal Chem 393: 171–175.
Bentley DRS, Balasubramanian HP, Swerdlow GP, Smith J, Milton CG,
Brown KP, Hall DJ, Evers CL, Barnes HR, Bignell JM, et al. 2008. Accurate
whole human genome sequencing using reversible terminator
chemistry. Nature 456: 53–59.
Blumenstiel JP, Noll AC, Griffiths JA, Perera AG, Walton KN, Gilliland WD,
Hawley RS, Staehling-Hampton K. 2009. Identification of EMS-induced
mutations in Drosophila melanogaster by whole-genome sequencing.
Genetics 182: 25–32.
Chen D, Ahlford A, Schnorrer F, Kalchhauser I, Fellner M, Viragh E, Kiss I,
Syvanen AC, Dickson BJ. 2008. High-resolution, high-throughput SNP
mapping in Drosophila melanogaster. Nat Methods 5: 323–329.
Cooper JL, Greene EA, Till BJ, Codomo CA, Wakimoto BT, Henikoff S. 2008.
Retentionofinduced mutations ina Drosophilareverse-genetic resource.
Genetics 180: 661–667.
Dahl F, Stenberg J, Fredriksson S, Welch K, Zhang M, Nilsson M, Bicknell D,
Bodmer WF, Davis RW, Ji H. 2007. Multigene amplification and
massively parallel sequencing for cancer mutation discovery. Proc Natl
Acad Sci 104: 9387–9392.
Deng J, Shoemaker R, Xie B, Gore A, LeProust EM, Antosiewicz-Bourget J,
Egli D, Maherali N, Park IH, Yu J, et al. 2009. Targeted bisulfite
sequencing reveals changes in DNA methylation associated with
nuclear reprogramming. Nat Biotechnol 27: 353–360.
Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W,
Fennell T, Giannoukos G, Fisher S, Russ C, et al. 2009. Solution hybrid
Mutation identification by genome targeted sequencing
selection with ultra-longoligonucleotides formassively parallel targeted
sequencing. Nat Biotechnol 27: 182–189.
Hazelrigg T, Kaufman TC. 1983. Revertants of dominant mutations
associated with the Antennapedia gene complex of Drosophila
melanogaster: Cytology and genetics. Genetics 105: 581–600.
Kent WJ. 2002. BLAT—the BLAST-like alignment tool. Genome Res 12: 656–
Lewis RA, Kaufman TC, Denell RE, Tallerico P. 1980. Genetic analysis of the
Antennapedia gene complex (Ant-C) and adjacent chromosomal
regions of Drosophila melanogaster. I. Polytene chromosome segments
84b-D. Genetics 95: 367–381.
Ley TJ, Mardis ER, Ding L, Fulton B, McLellan MD, Chen K, Dooling D,
Dunford-Shore BH, McGrath S, Hickenbotham M, et al. 2008. DNA
sequencing of a cytogenetically normal acute myeloid leukaemia
genome. Nature 456: 66–72.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G,
Abecasis G, Durbin R. 2009a. The sequence alignment/map (SAM)
format and SAMtools. Bioinformatics 25: 2078–2079.
Li JB, Gao Y, Aach J, Zhang K, Kryukov G, Xie B, Ahlford A, Yoon JK,
Rosenbaum AM, Zaranek AW, et al. 2009b. Multiplex padlock capture
and sequencing reveal human hypermutable CpG variations. Genome
Res 19: 1606–1615.
genetics. Trends Genet 24: 133–141.
Mardis ER. 2008b. Next-generation DNA sequencing methods. Annu Rev
Genomics Hum Genet 9: 387–402.
Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T,
Wong M, Bhattacharjee A, Eichler EE, et al. 2009. Targeted capture and
massively parallel sequencing of 12 human exomes. Nature 461: 272–
Okou DT, Locke AE, Steinberg KM, Hagen K, Athri P, Shetty AC, Patel V,
with the Illumina Genome Analyzer platform to sequence diploid target
regions. Ann Hum Genet 73: 502–513.
Platts AE, Land SJ, Chen L, Page GP, Rasouli P, Wang L, Lu X, Ruden DM.
2009. Massively parallel resequencing of the isogenic Drosophila
melanogaster strain w(1118); iso-2; iso-3 identifies hotspots for
mutations in sensory perception genes. Fly 3: 192–203.
Emig CJ, Dahl F, et al. 2007. Multiplex amplification of large sets of
human exons. Nat Methods 4: 931–936.
Sarin S, Prabhu S, O’Meara MM, Pe’er I, Hobert O. 2008. Caenorhabditis
elegans mutant allele identification by whole-genome sequencing. Nat
Methods 5: 865–867.
Shendure J, Ji H. 2008. Next-generation DNA sequencing. Nat Biotechnol 26:
Smith DR, Quinlan AR, Peckham HE, Makowsky K, Tao W, Woolf B, Shen L,
Donahue WF, Tusneem N, Stromberg MP, et al. 2008. Rapid whole-
genome mutational profiling using next-generation sequencing
technologies. Genome Res 18: 1638–1642.
Srivatsan A, Han Y, Peng J, Tehranchi AK, Gibbs R, Wang JD, Chen R. 2008.
High-precision, whole-genome sequencing of laboratory strains
facilitates genetic studies. PLoS Genet 4: e1000139. doi:
Wheeler DA, Srinivasan M, Egholm M, Shen Y, Chen L, McGuire A, He W,
Chen YJ, Makhijani V, Roth GT, et al. 2008. The complete genome of an
individual by massively parallel DNA sequencing. Nature 452: 872–876.
Winkler S, Schwabedissen A, Backasch D, Bokel C, Seidel C, Bonisch S,
Furthauer M, Kuhrs A, Cobreros L, Brand M, et al. 2005. Target-selected
mutant screen by TILLING in Drosophila. Genome Res 15: 718–723.
Zhai RG, Hiesinger PR, Koh TW, Verstreken P, Schulze KL, Cao Y, Jafar-Nejad
H, Norga KK, Pan H, Bayat V, et al. 2003. Mapping Drosophila mutations
with molecularly defined P element insertions. Proc Natl Acad Sci 100:
Received November 5, 2009; accepted in revised form March 24, 2010.
Wang et al.