Published online 8 November 2010 Nucleic Acids Research, 2011, Vol. 39, No.

2 e8
doi:10.1093/nar/gkq1005

Targeted resequencing of candidate genes

using selector probes
H. Johansson1,2, M. Isaksson1,2, E. Falk Sörqvist1,2, F. Roos1,2, J. Stenberg1,2,
T. Sjöblom1, J. Botling1, P. Micke1, K. Edlund1, S. Fredriksson3, H. Göransson Kultima4,
Olle Ericsson1,2,* and Mats Nilsson1,*
1
Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE 75185 Uppsala, 2Olink
Genomics, Dag Hammarskjölds väg 36B, SE 75183 Uppsala, 3Olink Biosciences, Dag Hammarskjölds väg 54A,
SE 75183 Uppsala and 4Department of Medical Sciences, Uppsala University, Akademiska Hospital, SE 75185

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Uppsala, Sweden

Received July 21, 2010; Revised September 22, 2010; Accepted October 7, 2010

ABSTRACT complete exomes, but as sequencing rapidly becomes

cheaper, exome sequencing can be expected to become
Targeted genome enrichment is a powerful tool for less rational. However, for many hypothesis-driven or
making use of the massive throughput of novel clinical investigations, it is more efficient and rational to
DNA-sequencing instruments. We herein present a direct sequencing to a small fraction of the genome, e.g. to
simple and scalable protocol for multiplex amplifi- a set of candidate genes. By limiting the field of search,
cation of target regions based on the Selector tech- more sequencing capacity can be spent to achieve high
nique. The updated version exhibits improved coverage of the region of interest (ROI). This is particu-
coverage and compatibility with next-generation- larly useful for investigations where deep sequencing is
sequencing (NGS) library-construction procedures required such as sequencing of mosaic tumor biopsies
for shotgun sequencing with NGS platforms. To and for clinical applications. Furthermore, it can also
demonstrate the performance of the technique, all facilitate analysis of large patient cohorts which is still
very expensive if performed on complete genomes.
501 exons from 28 genes frequently involved in
Several methods for enrichment of multiple target loci
cancer were enriched for and sequenced in speci- have been presented. The methods are in the most general
mens derived from cell lines and tumor biopsies. sense based on either hybridization capture or parallelized
DNA from both fresh frozen and formalin-fixed PCR amplification of multiple regions (5–17). For more
paraffin-embedded biopsies were analyzed and in-depth review of targeted resequencing methodologies,
94% specificity and 98% coverage of the targeted we refer to the reviews by Mamanova et al. (18) and
region was achieved. Reproducibility between repli- Turner et al. (19). Assays based on hybridization have
cates was high (R2 = 0, 98) and readily enabled de- proven to be effective in capturing the complete exome,
tection of copy-number variations. The procedure or a large fraction of it. However, relying solely on
can be carried out in <24 h and does not require hybridization for discrimination between target and
any dedicated instrumentation. non-target is difficult with regards to specificity and typic-
ally result in capture of 50–70% irrelevant sequences (6).
The problem is accentuated when capturing smaller
INTRODUCTION regions that require higher enrichment levels. If the ratio
between on- and off-target DNA is low, the risk of
DNA resequencing of whole mammalian genomes can be
misaligning off-target sequences to the target sequence
performed with a range of novel sequencing approaches
increases, which may lead to false positive, or even false
(1–4). Sufficient coverage to detect the vast majority of
negative results. This lack of specificity in the capture step
genetic variation in a complete genome can be achieved
can to some extent be counteracted in the sequencing
with a single instrument run which makes it a very
step by increasing the read length and/or performing
powerful tool for screening. As discussed below, there
paired end sequencing, however this comes at significant
are today also cost-efficient approaches for resequencing
increases in both cost and assay time. To allow for a more

*To whom correspondence should be addressed. Tel: +46 18 471 48 16; Fax: +46 18 471 48 08; Email: mats.nilsson@genpat.uu.se
Correspondence may also be addressed to Olle Ericsson. Tel: +46 18 495 31 22; Fax: +46 18 495 31 21; Email: olle.ericsson@olinkgenomics.com

The authors wish it to be known that, in their opinion, the first two authors and last two authors should be regarded as joint First Authors.

ß The Author(s) 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
e8 Nucleic Acids Research, 2011, Vol. 39, No. 2 PAGE 2 OF 13

general targeting of fragments, hybridization can be Swedish Biobank Legislation and Ethical Review Act
combined with an enzymatic discrimination and ampli- (reference 2006/325, Ethical review board in Uppsala).
fication step, a strategy which is successfully demon- The tissue samples were reviewed by a pathologist and
strated in PCR (20). Since it is difficult to perform highly only tumor tissues with a tumor cell fraction >50%
multiplex PCR reactions with high-success rate, massive were included. DNA was extracted from FFPE and
numbers of single-locus PCRs have to be applied, requiring frozen tissue sections using the QIAamp DNA Mini Kit
compartmentalization in small volumes to be cost- (Qiagen, Hamburg, Germany).
effective. This can be achieved by array based approaches
(9) or by emulsion PCR (10), both requiring sophisticated Design and oligonucleotides
liquid handling and amplification systems.
Two approaches have been presented for efficient Twenty-eight genes (Table 1) known to be mutated in lung
single-tube massively multiplexed amplification of and/or colon cancer were chosen for targeted
genomic loci. Both methods are based on ligase-assisted resequencing. A list of coding regions for each gene was

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DNA-circularization reactions that, similar to PCR, rely downloaded from the consensus coding sequence
on enzymatic specificity and dual hybridization recogni- database, CCDS (build 36.3) and a total number of 501
tion. Gap-fill padlock probes (11) are oligonucleotides regions covering 82 kb were targeted. Coding sequences
that hybridize with sequences flanking the ROI (e.g. were collected from hg18 (March, 2006 assembly), and
an exon). The gap between the ends of the probe is filled processed by the Disperse software (23) to generate a set
by a DNA polymerase and sealed by a DNA ligase, of restriction fragments using eight combinations of re-
creating a circle containing the targeted sequence. With striction enzymes. A subset of 1883 fragments
this approach, tens of thousands of probes have been (Supplementary Table S1) was selected based on length
amplified with very low enrichment of off-target (100–1000 nt), GC-content (20–65%), avoiding repetitive
material. However, the performance for exon capture genomic elements in the ends, and to achieve redundant
is less than ideal: at 322 mean coverage only 75% of coverage over targeted regions. In the current design, we
the bases were covered at >20 (12). The related aimed for a double-fragment redundancy on each targeted
selector probes template circularization of restriction
fragments and do not include synthetic DNA sequences
from the probe in the targeted sequence, thereby reducing Table 1. Genes included in the design with their corresponding
the fraction of irrelevant sequence in the captured DNA. number of exons and the total number of base pairs that was
A PCR-based version of the selector technique was used to aimed to be covered for each gene (ROI)
efficiently sequence the exons of 11 cancer genes (13) with Number Name Number Number Percentage
excellent specificity but with relatively high-amplification of exonsa of ROI bpa of ROI bp
bias and low coverage (70% coverage at >10 coverage at covereda
127 average coverage).
In order to retrieve useful sequence information from the 1 AKT3 14 1685 99
2 IDH1 8 1254 100
ROI, it is very important that the enrichment is unbiased, 3 HER4 28 4124 98
since insufficient or irreproducible coverage within the 4 CTNNB1 14 2472 100
target region will drastically lower the fraction of useful 5 PIK3CA 20 3260 100
sequence data obtained from an experiment. 6 FBXW7 13 2549 96
7 APC 15 8590 99
In this article, we present a non-PCR-based version 8 EGFR 30 4198 100
of the selector method (21), including a significantly 9 MET 20 4192 100
updated and optimized protocol as well as selector design 10 BRAF 18 2432 100
approach. Instead of using PCR an RCA-based multiple 11 CDKN2A 4 1074 100
12 PTEN 9 1359 98
displacement amplification (MDA) (22) is used, generating 13 CCND1 5 888 100
an amplification product which is easily integrated with 14 MRE11A 19 2431 99
shotgun library construction for short-read sequencing 15 ATM 62 9359 98
platforms. 16 KRAS 5 687 100
We evaluate the method for enrichment of a rela- 17 HER3 28 4357 98
18 AKT1 13 1594 94
tively small set of exons (501) with high coverage and 19 SMAD3 9 1352 100
relatively low-enrichment bias. Amplification of thou- 20 TP53 10 1330 100
sands of targeted fragments (1883) in one tube with 21 NF1 58 8865 98
high coverage is demonstrated in a simple process that 22 HER2 31 4357 95
23 SMAD2 10 1485 97
does not require genome centers to invest in additional 24 SMAD4 11 1778 100
instrumentation. 25 STK11b 9 1370 98
26 CCNE1 11 1357 100
27 AKT2 13 1597 100
MATERIALS AND METHODS 28 GNAS 14 2196 98

Preparation of tumor samples The last column shows the percentage of the ROI base pairs that were
covered in the design.
Matched fresh-frozen tumor/benign lung tissues and a
CCDS 14 April 2009.
b
FFPE tumor tissue were used in accordance with the No CCDS available, CDS used, 14 April 2009.
PAGE 3 OF 13 Nucleic Acids Research, 2011, Vol. 39, No. 2 e8

base to avoid the risk of fragment dropout due to muta- Following this step, to further remove non-specifically
tions in the restriction enzyme or probe-binding site. The bound DNA, the beads were washed in 1 M NaCl,
design achieved 99% coverage of targeted bases and the 10 mM Tris–HCl (pH 7.5), 5 mM EDTA and 0.1%
missing bases were found to be in or near repetitive Tween-20 in a total volume of 200 ml at 46 C for 30 min
elements. Selected restriction fragments are displayed with rotation.
in the supplementary gff-file. Selector probes serving
as template for circularization of each chosen fragment Circularization of targeted fragments
were designed using the ProbeMaker software (24). Each
selector consists of two Tm balanced sequences of 20–25 nt To circularize the genomic fragments, the beads were
complementary to the ends of its targeted restriction incubated in 1 Ampligase reaction buffer, 0.25 U/ml
fragment. Ampligase (Epicentre) and 0.1 mg/ml BSA in a total
The oligonucleotides (Integrated DNA Technologies) volume of 50 ml. The ligation mix was incubated at 55 C
hybridizing to 25 bps on each end of the targeted for 10 min.

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fragments where pooled in one tube. To add a 30 -biotin
to the probes the oligonucleotides where incubated in Amplification
2.5 mM total concentration with 1 Tdt buffer (NEB), To enrich for circular molecules, a MDA reaction
1 CoCl2 (NEB), 0.1 mM dUTP-biotin (Roche (Templiphi, GE life sciences) was carried out using the
Diagnostics) and 0.2 U/ml Terminal Transferase (NEB) circles as templates. The circularized molecules were
in a final volume of 50 ml. The reaction was incubated at separated from the beads into the solution by incuba-
37 C for 1 h and inactivated at 75 C for 20 min. To tion with 5 ml sample buffer at 95 C for 10 min and
remove unincorporated nucleotides the probes were thereafter collected by placing the tubes in a ring
purified on three consecutive G-50 columns (GE Life magnet rack and immediately aspirating the supernatant.
Sciences). To initiate MDA, 5 ml reaction buffer and 0.2 ml en-
zyme mix was added to the supernatant and incubated
Restriction digestion at 30 C for 4 h followed by deactivation at 65 C for
10 min.
Of each sample, 100 ng (200 ng for FFPE samples to
compensate for DNA fragmentation) was added to eight
different restriction reactions containing 1 unit each of Sequencing
two restriction enzymes and their corresponding compat- Of each MDA-amplification product, 300 ng was frag-
ible NEB buffer in 1 concentration and 0.85 mg/ml BSA mented using the Covaris S2 system and libraries where
in a total volume of 10 ml. The eight reactions were SfcI constructed from these using the SOLiD (v3) library con-
and Hpy188I in NEB buffer 4; DdeI and AluI in NEB struction kit (Applied Biosystems). The constructed
buffer 2; MseI and Bsu36I in NEB buffer 3; MslI and BfaI libraries where sequenced in barcoded sequencing reac-
in NEB buffer 4; HpyCH4III and Bsp1286 in NEB tions containing 20 different samples on a fourth of a
buffer 4; SfcI and NlaIII in NEB buffer 4; MseI and sequencing slide.
HpyCH4III in NEB buffer 4; HpyCH4V and EcoO109I
in NEB buffer 4 (New England Biolabs). The reactions Data analysis
were incubated at 37 C for 60 min followed by enzyme
deactivation at 80 C for 20 min. After this step the eight All analyses were made on the targeted 501 exonic regions
reactions were pooled. defined by CCDS. SOLiD (v3) sequence data from
four specimens, a Yoruba trio (NA1806-8, family Y009),
tumor material from a lung-cancer patient and two
Probe hybridization
pairs of matched cell lines derived from normal
The 80 ml of digested sample resulting from pooling the and breast cancer tissue, were all analyzed by
reactions were mixed with 10 pM individual concentration MosaikAligner version 1.0.1388 (Mosaik—The
of biotinylated selector probes (Integrated DNA MarthLab Available at: http://bioinformatics.bc.edu/
Technologies), 0.7 M NaCl, 7 mM Tris–HCl (pH 7.5), marthlab/Mosaik). MosaikAligner parameters were set
3.5 mM EDTA, and 0.07% Tween-20 in a total volume using jump database for mapping to the reference
of 160 ml. The solution was incubated at 95 C for 10 min, genome (hg18, mars 2006 assembly); -hs 15, -mm 6, -ms
75 C for 30 min, 68 C for 30 min, 62 C for 30 min, 55 C 6, -mhp 100 and -act 20. The alignment software was set to
for 30 min and 46 C for 10 h. allow six mismatches per 50-mer read.
Targeted amplification specificity for all samples was
Solid phase capture and wash calculated as the proportion of reads that uniquely
aligned to the genome mapped to the targeted region
To remove off-target material, the hybridization solu- (Table 2).
tion was mixed with 10 ml M-280 streptavidin coated
magnetic beads (3.35  107 beads/ml; Invitrogen) in
SNP array analysis
0.7 M NaCl, 7 mM Tris–HCl (pH 7.5), 3.5 mM EDTA
and 0.07% Tween-20 in a final volume of 200 ml, and SNP-array experiments were performed according to the
incubated at room temperature for 15 min. After incuba- standard protocols for Affymetrix GeneChipÕ Mapping
tion, the beads were collected using a ring magnet. NspI-250K arrays (Gene Chip Mapping 500K Assay
e8 Nucleic Acids Research, 2011, Vol. 39, No. 2 PAGE 4 OF 13

Table 2. Sequencing results for each sample showing the total number of reads obtained for each sample and the percentage of them that align
to the human genome build hg18, and the percentage of the hg18 uniquely aligned reads that aligns to the specified region

Sample Total hg18 Amplified Region Region of

number region of interest interest ±50 bp
of reads
Percentage Percentage Percentage Percentage Percentage
of all readsa of unique of unique of unique of unique
readsb readsc readsc readsc

NA18506 1653229 51.34 40.02 92.23 66.21 81.41

NA18507 1709623 50.43 40.16 93.31 67.72 82.99
NA18508 1187613 48.25 38.07 92.82 67.62 82.97
HCC1143 Normal 1687199 51.26 41.23 93.45 67.03 82.38
HCC1143 Tumor 1452999 52.86 43.21 93.85 65.80 81.69
HCC1599 Normal 2617647 53.72 42.48 92.20 65.20 80.45

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HCC1599 Tumor 1882203 49.44 41.50 95.61 68.70 85.75
Normal tissue fresh frozen 1567773 53.47 44.27 94.63 67.98 83.42
Tumor tissue fresh frozen 1841970 51.17 42.06 94.32 67.30 83.33
Tumor tissue FFPE 1878918 45.79 37.23 93.95 74.11 88.66
NA18506 2008052 49.63 39.41 92.93 66.98 82.07
All samples 19487226 50.76 40.93 93.55 67.62 83.12
a
Percentage of the total number of reads that aligned to the human genome and selector-induced sequences, counting all reads that align (those that
align more than one time is randomly placed and counted only once).
b
Percentage of the total number of reads that aligned uniquely to hg18.
c
Percentage of the uniquely aligned reads to hg18 that aligned uniquely to the region specified.

Manual (P/N 701930 Rev2.), Affymetrix Inc., Santa Analysis of input DNA amount requirement
Clara, CA, USA) and the arrays were scanned using the A titration of different amount of DNA from 100 to
GeneChipÕ Scanner 3000 7G. Normal samples analyzed 1600 ng DNA (combined for all eight restriction diges-
at Uppsala Array Platform were used as a reference set to tions) was subjected to our enrichment protocol. The
produce log ratios. The rank segmentation algorithm, amplified DNA was sent to GATC Biotech (Constance,
similar to the Circular Binary Segmentation (CBS) algo- Germany) for Illumina GAII sequencing. The 32-mer
rithm (25), in the software Nexus from Biodiscovery was reads were mapped to our amplified region as described
used to segment the data across the genome. The signifi- above, but only allowing for four mismatches (-mm 4).
cance threshold for segmentation was set at 1  106, also The specificity was calculated as the fraction of unique
requiring a minimum of 40 probes per segment. The reads that mapped to our amplified region.
results are shown as segmented log2 ratio in Figure 4a.

Copy-number analysis RESULTS

We designed a somatic mutation detection assay for 28
Copy-number alterations were detected by analyzing the
genes frequently mutated in solid tumors such as colon,
difference in sequencing depth in each of the targeted pos-
lung and breast cancer (Table 1), targeting all 501 coding
itions with 20 sequencing depth or more in the normal
regions according to the CCDS database, defining our
DNA sample for respective tumor–normal pair. Reference
ROI. The new version of the selector technique is
allele ratios were calculated by dividing reference allele call
described in detail in Figure 1a and in brief involves
frequency by the total sequencing depth for each position
solid-phase enrichment and probe-templated circulariza-
in the targeted region.
tion of specific-restriction fragments that are amplified
To enable comparison of the sequencing and the
using a circle-specific RCA-based MDA. The overall
GeneChip data (Figure 4b), the sequencing depth log2
cost and quality of a targeted-sequencing experiment are
ratios were derived from the difference in mean hits per
determined by the performance of the enrichment tech-
gene for the normal–tumor pair. They were based on pos- nique. Important metrics to consider are in silico design
itions within the amplified ROI with a sequencing depth of coverage, actual coverage, specificity, reproducibility and
at least 20 in the normal DNA sample. enrichment uniformity across the targeted loci. To
evaluate these parameters, we sequenced a HapMap trio
(NA18506, NA18507 and NA18508) using a SOLiD (v3)
Length dependency analysis
instrument. The sequencing libraries were bar-coded to
To extract junction-specific reads, a library where two allow sequencing of 20 samples on a fourth of a slide.
copies of each selector were concatenated to each other On average, 899 000 reads mapped to the genome, and
was created. The reads were aligned to the library and the of the uniquely mapping reads, 94% (range 92–96%)
reads aligning uniquely over the junction were used as mapped to the amplified region, demonstrating the high
junction specific reads. specificity of this approach corresponding to an
PAGE 5 OF 13 Nucleic Acids Research, 2011, Vol. 39, No. 2 e8

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Figure 1. Selector probe technology and performance. (a) Overview of the selector-probe amplification procedure. A ROI (e.g. an exon) is targeted
by probing several redundant fragments (dark blue) selected from eight separate restriction digestion reactions of a genomic DNA sample (light
blue). The restriction-digested DNA samples are pooled and hybridized with biotin-tagged selector probes complementary to the ends of each
targeted restriction fragment (red). The targeted fragments are then captured on streptavidin-coated magnetic beads. The fragments are circularized
by DNA ligation after removal of non-targeted DNA. Finally, the circularized fragments are released from the beads and specifically amplified using
the RCA-based MDA. (b) Correlation of the relative coverage of individual bases between two replicate enrichment and sequencing experiments.
(c) The cumulative fraction of the target sequence covered is plotted as a function of different relative read-depths, indexed to the mean coverage, in
three sequencing experiments using DNA from the NA18506 HapMap cell line (black line), fresh-frozen lung cancer tissue (blue broken line) and
FFPE lung cancer tissue (red broken line).

enrichment factor [=(sequenced bp in ROI/sequenced bp Table 3. Specificity values obtained for experiments with different
off ROI)/(size of ROI/size of the genome)] of 200 000 amount of input DNA
(enrichment statistics for all samples are summarized in
Input DNA (ng) Specificitya (%)
Table 2). Some amplified fragments will also contain
adjacent off ROI sequence. With a strict coding-sequence 1600 96.0
target definition, 68% of the reads mapped on target, and 800 91.0
83% on target ±50 bp. The reproducibility in coverage 400 83.9
200 75.0
between samples is illustrated in Figure 1b and was 100 66.0
calculated to 0.98 (R2, linear regression). Furthermore,
98% of the targeted bases were covered at >10% of the a
Percentage of the uniquely aligned reads to hg18 that aligned uniquely
mean base coverage (mean coverage = 273, Figure 1c and to the region specified.
Supplementary Figure S1). To investigate how much input
DNA is required for the method, we performed
enrichment experiments on different amounts of It is of great importance that an enrichment tech-
template DNA (Table 3). We observed that the specificity nique introduces minimal distortion of the original
decreased when <800-ng DNA was added to the reaction, allele ratios in the analyzed sample. To assess the
and therefore we chose not to use less DNA than 800 ng ability of the presented Selector protocol to preserve
per sample in this study. original allele ratios, we sequenced a HapMap trio
e8 Nucleic Acids Research, 2011, Vol. 39, No. 2 PAGE 6 OF 13

(NA18506, NA18506 and NA18508) and compared tumor and matched control samples of two breast-cancer
our calls with results made available within the HapMap cell lines (HCC1143 and HCC1599). By comparing the
project (26). Reference allele-frequency analysis was variant calls from the tumor and the matched normal
performed using MosaikAligner and the result was tissue, it is possible to distinguish germline variants from
compared to the genotypes of the 164 available SNPs in somatic mutations. The results from comparing allele
the target sequence in the HapMap database which ratios are plotted in the lower panels of Figure 3a and b
overlapped with the ROI (Figure 2 and Table 4). The and the calls are summarized in Table 5. The difference in
concordance was 100% for covered SNPs within ROI allele ratio between the tumor and normal cell line was
and 99% including targeted SNPs outside of ROI >0.3 in 14 ROI positions in HCC1143, and 7 ROI positions
(in total 383). in HCC1599. The differences in HCC1143 were due
To investigate the utility of this enrichment technique to loss-of-heterozygosity (LOH) in eight genes, caused by
for analysis of somatic mutations in cancer, we sequenced allelic amplification (CCND1), deletion [MET, CDKN2A,
MRE11A, ATM, NF1 and ERBB2 (HER2)] and copy-

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number neutral LOH (uniparental disomy, APC). An
(A!G) mutation was found in the HER3 gene outside
the ROI. The allele-ratio differences in HCC1599 were
due to LOH in five genes, caused by allelic amplification
(CCNE1) and deletions (APC, EGFR, TP53 and NF1).
Finally, one (T!A) mutation in the TP53 gene was
identified just outside the ROI in association with loss of
the normal allele, which confirms a previous study of
the somatic mutation spectrum in this cell line (27). In
addition to mutation detection, we also compared the
relative read depth at different positions in the ROI to
detect copy-number alterations. Comparison of relative
read depth at each position in the ROI in the cell
line pairs HCC1143/HCC1143BL and HCC11599/
HCC11599BL are plotted in upper panels in Figure 3a
and b, respectively. To exclude that the differences in
coverage originated from poor reproducibility of the
assay, we also analyzed a replicate sequencing experiment
of the NA18506 DNA sample in the same manner
(Figure 3c). The analysis revealed considerable relative
copy-number variation between the tumor and normal
cell line, including amplification, duplication or deletion
of the majority of investigated genes.
Finally, we evaluated the technique on DNA prepared
from normal and tumor tissue from a lung-cancer patient.
We applied the same subtractive approach to analyze the
mutational status of the 28 genes as with the cancer cell
line samples. We detected copy-number deviations that
confirmed previous microarray analysis performed using
an Affymetrix 250K SNP chip (Figure 4a, Supplementary
Figure S2). The deviations were smaller in this experi-
ment than for the cell lines, which is consistent with a
tumor-cell content of 50% in this sample. The correl-
ation between the array-based copy-number estimate
and our sequencing-based estimate proved to be strong
(R2 = 0.9999) (Figure 4b). We detected a single base-pair
deletion that had previously been identified in the TP53
gene by Sanger sequencing (28) (Figure 4c). The majority
of patient tumor samples are prepared and stored as
formalin-fixed paraffin embedded (FFPE) specimens.
The potential utility of a somatic mutation analysis
Figure 2. Analysis of concordance with available SNP genotypes of the technique is therefore greatly enhanced if it is compat-
(a) NA18506; (b) NA18507; and (c) NA18508 DNA samples. The allele ible with DNA from FFPE tissue samples, which are
ratio of all positions where the SNP genotype is available in the often severely degraded. To evaluate this potential we
HapMap database are plotted as a function of read depth. Green amplified and sequenced the 28 genes using DNA from
dots represent homozygous SNP positions in the database and red
dots represent heterozygous positions. Heterozygous positions were an FFPE sample from the same lung cancer patient.
defined as having an allelic ratio of 0.35 and 0.85 (dashed horizontal We found the TP53 deletion mutation also in this
lines). The vertical dashed line indicates a read depth of 20. material (Figure 4d). By quantifying the reads mapping
PAGE 7 OF 13 Nucleic Acids Research, 2011, Vol. 39, No. 2 e8

Table 4. SNP concordance with data available from three HapMap samples (NA18506, NA18507 and NA18508) at two coverage thresholds:
covered at least once and covered at least 20 times

Sample Relation Coverage Region Number Covered (%) Homozygoteb Heterozygote Concordance (%)
of SNPsa
Selectors Hapmap Selectors Hapmap

NA18506 Son 1 ROI 165 100.00 161 161 4 4 100.00

Amplified region 382 99.21 346 347 33 32 99.74
20 ROI 165 91.52 147 147 4 4 100.00
Amplified region 382 75.39 265 265 23 23 100.00
NA18507 Father 1 ROI 164 100.00 151 151 13 13 100.00
Amplified region 382 99.21 330 326 49 53 98.42
20 ROI 164 92.07 138 138 13 13 100.00
Amplified region 382 74.87 248 249 38 37 99.65
NA18508 Mother 1 ROI 165 100.00 151 151 14 14 100.00

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Amplified region 383 98.96 334 329 45 50 98.68
20 ROI 165 80.61 122 122 11 11 100.00
Amplified region 383 62.92 211 211 30 30 100.00

Positions are considered as heterozygote if the reference allele ratio is between 0.35 and 0.85.
a
Number of HapMap SNPs overlaping with region. NN positions omitted.
b
Reference allele ratio under 0.35 or above 0.85.

to junctions of the circularized fragments, we were able difficult to assess whether MDA introduce less or more
to retrieve information about the performance of indi- amplification bias compared to PCR, because of
vidual selectors. We observed worse representation the probe redundancy and since different sequencing
of the longer fragments than the shorter in the FFPE platforms were used to sequence the PCR amplicons
sample compared to the fresh frozen (Figure 5), resulting (454, long read) and the MDA products (SOLiD, short
in more uneven amplification (Figure 1c). However, read). In contrast to PCR, the MDA amplification gener-
the difference in coverage between the shortest and the ates a double-stranded high-molecular weight amplifi-
longest fragments in the range 100–300 bp was less than cation product that is very similar in nature to genomic
a factor of two, so with a different probe design targeting DNA. We have demonstrated compatibility with the
shorter fragments, evenness of coverage should be standard sample preparation procedures for both SOLiD
improved. and Illumina short-read instruments. In contrast, to be
compatible with the short-read-sequencing platforms, a
PCR product requires concatemerization by ligation,
DISCUSSION followed by fragmentation, which is a more complicated
In this article, we report on a method for targeted enrich- scheme. For emulsion PCR, an additional fragmentation
ment of a relatively small subset of genes that produces step is also required before the PCR resulting in a very
the targeted regions in an unbiased fashion with very low laborious procedure. The MDA also avoids clonal propa-
amount of off-target material. For a small set of samples, gation of polymerization artifacts by employing rolling
it would be possible to sequence the same region with circle amplification, a feature valuable for detection of
PCR and Sanger sequencing with an estimated 384-well rare variants.
plate of reactions for each sample. We are however In addition to the importance of maintaining high
convinced that the proposed scheme is more practical specificity, only enriching regions that are targeted, it is
also for small collections of samples. also important to minimize the number of near-target base
Compared to the previously published version of pairs that are amplified. The restriction enzyme-based
the selector technology (13,29), the current protocol approach of the selector technology provides very high-
uses MDA instead of PCR. By eliminating the PCR on-target rate by design, between 50–70% of the
primers in the probes, the oligonucleotide length, and amplicons are on exons in the majority of designs (30).
thereby also cost, is significantly reduced. Probe cost This is comparable and even slightly better than published
has further been reduced through the introduction of work using parallel PCR on the Raindance platform (10).
a solid-phase purification step to remove excess probe In this article exons with an average size of 167 bp where
molecules, replacing the enzymatic degradation used in targeted with amplicons of between 300 and 600 bp, which
the previous protocol, requiring expensive uracil residues will generate a significant proportion of near-exon
in the probes. The invasive cleavage that was used to sequence. The selector amplicons range between 100 and
generate the majority of fragments in the previous 1000 and can more easily be adjusted to fit the target
protocol was avoided in this version, since we observed length when selecting from eight different restriction
worse bias when it was used (data not shown). Thus enzyme reactions (30).
in this study, we only used restriction digestion to Multiplex targeted sequencing is of particular interest
generate the fragments. We have further increased for molecular cancer diagnostics as genetic aberra-
design redundancy, which should improve coverage. It is tions in growth-factor-signaling pathways can be decisive
e8 Nucleic Acids Research, 2011, Vol. 39, No. 2 PAGE 8 OF 13

for treatment with certain novel drugs. When several

genes have to be analyzed separately in an old pipeline
using PCR followed by Sanger sequencing, the diag-
nostic process becomes time-consuming and costly.
Also, as tumor material often is limited, e.g. in the
form of small biopsies, it is difficult to obtain
sufficient DNA for a multitude of separate targeted-
mutation assays. The current selector protocol con-
sumes only 800 ng of genomic DNA. We believe that
this method can be useful for clinical diagnostics since it
can be applied on routinely collected FFPE samples.
The Selector probe set used in this study enriched
for nearly all bases within the ROI except for only 1.4%

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(30 regions) of the bases being unavailable applying the
current design criteria. When using targeted sequencing
for diagnostic purposes, it is often important to be
able to analyze all bases within the ROI that can be
uniquely mapped with the read length produced by the
sequencing instrument. The bases that we failed to cover
with selected fragments either contained repetitive
elements or SNPs in the hybridization sites. To achieve
complete coverage, manual examination of the failed
regions enables us to allow some fragments back into
the design that have been previously rejected by the
restrictive design filters.
Targeted resequencing is expected to be one of the
major applications for next-generation sequencing. The
current high-throughput platforms are in some aspects
suboptimal for targeted resequencing. To meet the
demand of quick and simple sequencing, several scaled
down sequencing platforms have been announced (e.g.
454 GS Junior, iScan and Ion Torrent), and to harness
the power of these instruments, simple, efficient and
high-quality enrichment technologies are needed. We
present a technology that offers high-performance
targeted analyses using a simple and scalable protocol.
To our knowledge, this is the only procedure for
scalable multiplex enrichment of targets that achieves
high (>95%) coverage and high (>95%) specificity
without introducing requirements of dedicated instrumen-
tation. Versions of the technology enabling introduction
of sequencing primers and barcodes during amplification
are under development. So far, direct library free
amplicon sequencing has been limited to the 454
sequencing platform, however, development of existing
and new platforms will make this an attractive option
for several sequencing technologies.

tumor-derived cell line (HCC1143/HCC1599) and then divided with

the sum of called bases for that position in the two samples. The
exons of the 28 genes are concatenated in the order specified in
Table 1 and gene shifts are demarked by alternating background
Figure 3. Somatic mutation analysis of two breast cancer cell lines with color. The lower panels show the value obtained from each base
matched normal controls HCC1143/HCC1143BL (a) and HCC1599/ when the allelic ratio between the major and minor allele of the
HCC1599BL (b). In each position in the targeted region, the number normal sample is subtracted from the corresponding allelic ratio of
of bases called in the matched normal cell line (HCC1143BL/ the tumor sample. (c) Base-by-base comparison of two replicate
HCC1599BL) is subtracted from the number of bases called in the sequencing experiments of the cell line NA18506.
Table 5. Potential somatic mutations are listed as defined as base positions with an absolute difference in reference allele ratio of at least 0.30 between tumor and normal samples

Sample Chr (hg18) Position Gene Reference Normal Tumor T.Ratio  Call Location
(hg18) sequence N.Ratioa
Ratio A G C T Ratio A G C T
PAGE 9 OF 13

HCC1143 NC_000005.8 112190753 APC T 0.38 0 0 52 32 1.00 0 0 0 78 0.62 LOH, CNN ROIb
HCC1143 NC_000005.8 112203669 APC G 0.57 65 85 0 0 1.00 0 160 0 0 0.43 LOH, CNN ROIb
HCC1143 NC_000005.8 112204224 APC G 0.61 189 292 0 0 1.00 3 597 0 0 0.39 LOH, CNN ROIb
HCC1143 NC_000005.8 112205070 APC G 0.61 43 68 0 0 1.00 0 144 0 0 0.39 LOH, CNN ROIb
HCC1143 NC_000007.12 116126908 MET C 0.62 0 0 114 69 0.00 0 0 0 43 0.62 LOH ROIb
HCC1143 NC_000007.12 116127498 MET A 0.74 62 22 0 0 0.08 2 21 0 0 0.65 LOH ROIb
HCC1143 NC_000007.12 116223258 MET G 0.57 116 152 0 0 0.99 1 114 0 0 0.42 LOH ROIb
HCC1143 NC_000007.12 116223333 MET G 0.59 75 136 0 0 1.00 0 89 0 0 0.41 LOH ROIb
HCC1143 NC_000009.10 21961039 CDKN2A G 1.00 0 28 0 0 0.00 0 0 0 0 1 ROIb
HCC1143 NC_000011.8 69172091 CCND1 G 0.49 70 68 0 0 0.08 531 146 0 0 0.41 LOH, AMP ROIb
HCC1143 NC_000011.8 93865568 MRE11A C 0.63 0 0 20 12 1.00 0 0 27 0 0.38 LOH ROIb
HCC1143 NC_000011.8 107668697 ATM C 0.66 0 0 193 100 0.05 0 0 5 87 0.60 LOH ROIb
HCC1143 NC_000017.9 26577611 NF1 G 0.7 12 28 0 0 0.00 14 0 0 0 0.70 LOH ROIb
HCC1143 NC_000017.9 35137563 HER2 C 0.75 0 6 18 0 0.00 0 27 0 0 0.75 LOH ROIb
HCC1599 NC_000005.8 112182868 APC C 0.68 0 2 393 179 0.15 0 0 32 179 0.53 LOH ROIb
HCC1599 NC_000005.8 112203516 APC T 0.62 85 0 0 136 0.99 1 0 0 86 0.37 LOH ROIb
HCC1599 NC_000005.8 112206694 APC G 0.56 147 187 1 0 0.03 117 4 0 0 0.53 LOH ROIb
HCC1599 NC_000007.12 55181842 EGFR C 0.67 1 0 177 87 0.99 0 0 155 2 0.32 LOH ROIb
HCC1599 NC_000017.9 7520197 TP53 G 0.57 0 26 19 1 1.00 0 10 0 0 0.43 LOH ROIb
HCC1599 NC_000017.9 26610220 NF1 A 0.48 29 31 0 0 0.85 23 4 0 0 0.37 LOH ROIb
HCC1599 NC_000019.8 35006506 CCNE1 C 0.65 0 0 108 57 0.16 0 0 75 401 0.50 AMP ROIb
HCC1143 NC_000002.10 212252269 HER4 G 0.52 14 15 0 0 0.21 19 5 0 0 0.31 Amp. Regionc
HCC1143 NC_000007.12 116185999 MET C 0.62 0 0 34 21 0.05 0 0 1 20 0.57 LOH Amp. Regionc
HCC1143 NC_000011.8 93851696 MRE11A C 0.69 0 0 27 12 1.00 0 0 23 0 0.31 LOH Amp. Regionc
HCC1143 NC_000011.8 93865455 MRE11A C 0.65 0 0 28 15 0.96 0 0 23 1 0.31 LOH Amp. Regionc
HCC1143 NC_000012.10 54763961 HER3 A 0.58 131 0 0 96 0.00 0 0 0 122 0.58 LOH Amp. Regionc
HCC1143 NC_000012.10 54764761 HER3 T 0.97 0 0 1 32 0.62 0 0 3 5 0.34 Amp. Regionc
HCC1143 NC_000012.10 54766850 HER3 G 0.46 21 18 0 0 0.00 22 0 0 0 0.46 LOH Amp. Regionc
HCC1143 NC_000012.10 54768447 HER3 T 0.68 0 15 0 32 0.06 0 16 0 1 0.62 LOH Amp. Regionc
HCC1143 NC_000012.10 54778054 HER3 A 1.00 195 0 0 0 0.05 3 60 0 0 0.95 A ! G Amp. Regionc
HCC1143 NC_000017.9 26510278 NF1 G 0.48 24 22 0 0 1.00 0 49 0 0 0.52 LOH, CNN Amp. Regionc
HCC1143 NC_000017.9 26584058 NF1 C 0.65 32 0 59 0 0.10 47 0 5 0 0.55 LOH, CNN Amp. Regionc
HCC1143 NC_000017.9 26584383 NF1 G 0.64 11 21 0 0 1.00 0 40 0 0 0.36 LOH, CNN Amp. Regionc
HCC1143 NC_000017.9 26677419 NF1 T 0.7 0 0 25 57 1.00 0 0 0 96 0.30 LOH, CNN Amp. Regionc
HCC1143 NC_000017.9 26679002 NF1 T 0.6 81 4 0 129 1.00 0 0 0 225 0.40 LOH, CNN Amp. Regionc
HCC1143 NC_000017.9 35119531 HER2 C 0.53 0 0 75 66 0.00 0 0 0 145 0.53 LOH, CNN Amp. Regionc
HCC1143 NC_000017.9 35122241 HER2 C 0.6 0 0 36 24 0.02 0 0 1 43 0.58 LOH, CNN Amp. Regionc
HCC1599 NC_000001.9 241867813 AKT3 T 0.67 0 0 10 20 0.33 0 0 18 9 0.33 Amp. Regionc
HCC1599 NC_000002.10 212134711 HER4 A 0.59 24 0 17 0 0.13 1 0 7 0 0.46 LOH Amp. Regionc
HCC1599 NC_000002.10 212192049 HER4 T 0.67 0 0 7 14 1.00 0 0 0 2 0.33 Amp. Regionc
HCC1599 NC_000002.10 212252169 HER4 A 0.56 84 65 0 0 0.14 8 48 0 0 0.42 LOH Amp. Regionc
HCC1599 NC_000002.10 212252269 HER4 G 0.71 10 24 0 0 0.00 10 0 0 0 0.71 LOH Amp. Regionc
HCC1599 NC_000002.10 212323573 HER4 C 0.67 0 0 250 122 0.98 0 0 208 4 0.31 LOH Amp. Regionc
HCC1599 NC_000002.10 212323761 HER4 T 0.63 0 0 39 66 0.29 0 0 25 10 0.34 LOH Amp. Regionc
HCC1599 NC_000004.10 153464702 FBXW7 C 0.43 0 12 9 0 0.00 0 21 0 0 0.43 LOH Amp. Regionc
HCC1599 NC_000004.10 153551830 FBXW7 T 0.61 0 11 0 17 1.00 0 0 0 26 0.39 LOH Amp. Regionc
HCC1599 NC_000007.12 55182141 EGFR G 0.45 0 14 0 17 1.00 0 8 0 0 0.55 LOH Amp. Regionc
HCC1599 NC_000007.12 55187671 EGFR A 0.66 61 32 0 0 0.97 57 2 0 0 0.31 LOH Amp. Regionc
Nucleic Acids Research, 2011, Vol. 39, No. 2

HCC1599 NC_000007.12 55196682 EGFR G 0.55 94 116 0 0 1.00 0 97 0 0 0.45 LOH Amp. Regionc
(continued)
e8

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Table 5. Continued

Sample Chr (hg18) Position Gene Reference Normal Tumor T.Ratio  Call Location
(hg18) sequence N.Ratioa
Ratio A G C T Ratio A G C T

HCC1599 NC_000007.12 55227257 EGFR A 0.66 78 41 0 0 0.03 1 28 0 0 0.62 LOH Amp. Regionc
HCC1599 NC_000007.12 55240320 EGFR G 0.77 17 56 0 0 0.00 9 0 0 0 0.77 LOH Amp. Regionc
HCC1599 NC_000012.10 25269723 KRAS T 0.76 0 0 6 19 0.00 0 0 4 0 0.76 Amp. Regionc
HCC1599 NC_000012.10 54764729 HER3 A 1.00 20 0 0 0 0.67 2 1 0 0 0.33 Amp. Regionc
HCC1599 NC_000012.10 54764863 HER3 T 1.00 0 0 0 25 0.60 0 0 2 3 0.40 Amp. Regionc
HCC1599 NC_000012.10 54766915 HER3 G 0.7 17 40 0 0 0.33 24 12 0 0 0.37 Amp. Regionc
e8 Nucleic Acids Research, 2011, Vol. 39, No. 2

HCC1599 NC_000012.10 54780089 HER3 A 0.67 65 0 32 0 0.34 22 0 42 0 0.33 Amp. Regionc

HCC1599 NC_000014.7 104310237 AKT1 T 0.65 0 0 36 68 1.00 0 0 0 49 0.35 LOH Amp. Regionc
HCC1599 NC_000014.7 104329938 AKT1 A 0.51 69 66 0 0 1.00 51 0 0 0 0.49 LOH Amp. Regionc
HCC1599 NC_000015.8 65244539 SMAD3 G 0.63 0 98 56 0 0.99 0 124 1 0 0.36 LOH Amp. Regionc
HCC1599 NC_000015.8 65244934 SMAD3 C 0.67 8 0 16 0 1.00 0 0 14 0 0.33 LOH Amp. Regionc
HCC1599 NC_000015.8 65264504 SMAD3 C 0.64 0 0 32 18 0.97 0 0 37 1 0.33 LOH Amp. Regionc
HCC1599 NC_000017.9 7518335 TP53 T 1.00 0 0 0 48 0.10 9 0 0 1 0.90 T ! A Amp. Regionb
HCC1599 NC_000019.8 1157823 STK11 A 1.00 25 0 0 0 0.67 4 0 0 1 0.33 Amp. Regionc
HCC1599 NC_000019.8 1158238 STK11 G 0.63 0 153 0 90 1.00 0 65 0 0 0.37 LOH Amp. Regionc
HCC1599 NC_000019.8 1158280 STK11 C 0.58 0 0 188 134 0.98 0 0 91 2 0.39 LOH Amp. Regionc
HCC1599 NC_000019.8 1169523 STK11 G 0.62 0 39 0 24 1.00 0 30 0 0 0.38 LOH Amp. Regionc
HCC1599 NC_000019.8 1170274 STK11 G 0.55 13 16 0 0 1.00 0 9 0 0 0.45 LOH Amp. Regionc
HCC1599 NC_000019.8 1177772 STK11 C 0.49 0 0 23 24 1.00 0 0 9 0 0.51 LOH Amp. Regionc
HCC1599 NC_000019.8 1177901 STK11 G 0.33 0 11 0 22 1.00 0 6 0 0 0.67 LOH Amp. Regionc
HCC1599 NC_000019.8 35005416 CCNE1 C 0.77 0 0 20 6 0.13 0 0 3 20 0.64 Amp. Regionc
HCC1599 NC_000019.8 35005443 CCNE1 C 0.48 1 12 12 0 0.08 0 66 6 0 0.40 LOH Amp. Regionc
HCC1599 NC_000019.8 45435698 AKT2 G 0.7 25 58 0 0 0.22 169 49 0 0 0.48 LOH, AMP Amp. Regionc

The number of respective base call is shown for both normal and tumor sample, the reference allele ratios, the reference allele ratio and our interpretation for the probable cause for the ratio
difference are listed for all positions.
a
Only base positions with an absolute ratio difference above 0.3 between tumor and normal are listed.
b
The position is located within the region of interest.
c
The position is located within the amplified region but not in the region of interest.
PAGE 10 OF 13

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PAGE 11 OF 13 Nucleic Acids Research, 2011, Vol. 39, No. 2 e8

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Figure 4. (a) Mutation detection and CNV analysis of a lung cancer patient sample with matched control. Upper panel: for each position in the
targeted region the number of bases called in the patient-matched normal tissue is subtracted from the number of bases called in the tumor-derived
DNA, and then divided with the sum of called bases for that position in the two samples. The exons of the 28 genes are lined up after each other and
genes are demarked by alternating background color. Middle panel: the inferred gene copy-number variation in the corresponding genomic loci
illustrated by log2 ratios (pink line) derived from SNP array data (Affymetrix Gene Chip Mapping 250K arrays). Middle panel: the log2 ratio (pink
line) of the copy-number analysis done on an Affymetrix micro array. Lower panel: the allelic ratio between the major and minor allele at each
position is compared between the two samples by subtraction (b). A correlation plot between the Affymetrix Gene Chip log2 tumor/normal signal
ratio and the log2 tumor/normal sequencing read depth ratio. (c) Detection of a single base pair deletion in the TP53 gene. Forward (brown) and
reverse (blue) reads are aligned to a 15-bp region of the TP53 gene. Deleted bases are indicated by dashed lines. Alignment visualized in Integrative
Genomic Viewer (IGV ver.1.4.2). (d) Detection of the same mutation in the FFPE sample from the same tumor.
e8 Nucleic Acids Research, 2011, Vol. 39, No. 2 PAGE 12 OF 13

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