Paired-end sequencing of Fosmid libraries by Illumina
Louise J.S. Williams, Diana G. Tabbaa, Na Li,1Aaron M. Berlin, Terrance P. Shea,
Iain MacCallum, Michael S. Lawrence, Yotam Drier, Gad Getz, Sarah K. Young,
David B. Jaffe, Chad Nusbaum, and Andreas Gnirke2
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02141, USA
Eliminating the bacterial cloning step has been a major factor in the vastly improved efficiency of massively parallel
sequencing approaches. However, this also has made it a technical challenge to produce the modern equivalent of the
Fosmid- or BAC-end sequences that were crucial for assembling and analyzing complex genomes during the Sanger-based
sequencing era. To close this technology gap, we developed Fosill, a method for converting Fosmids to Illumina-compatible
jumping libraries. We constructed Fosmid libraries in vectors with Illumina primer sequences and specific nicking sites
flanking the cloning site. Our family of pFosill vectors allows multiplex Fosmid cloning of end-tagged genomic fragments
bulk of each cloned insert, we introduced two nicks in the vector, translated them into the inserts, and cleaved them.
Recircularization of the vector via coligation of insert termini followed by inverse PCR generates a jumping library for
of short, incorrectly spaced and chimeric artifacts sufficiently low, to enable applications such as mapping of structural
variation and scaffolding of de novo assemblies. We demonstrate the power of Fosill to map genome rearrangements in
a cancer cell line and identified three fusion genes that were corroborated by RNA-seq data. Our Fosill-powered assembly
of the mouse genome has an N50 scaffold length of 17.0 Mb, rivaling the connectivity (16.9 Mb) of the Sanger-sequencing
based draft assembly.
[Supplemental material is available for this article.]
Paired-end sequencing of large DNA fragments cloned in Fosmid
(Kim et al. 1992) or BAC (Shizuya et al. 1992) vectors were a
mainstay of genome projects during the Sanger-based sequencing
era. The large spans, particularly of BAC ends, helped resolve long
repeats and segmental duplications and provided long-range
connectivity in shotgun assemblies of complex genomes (Adams
et al. 2000; Venter et al. 2001; Waterston et al. 2002). Fosmids are
shorter than BACs but much easier to generate. Their consistent,
narrow insert-size distribution centered around 35–40 kb enabled
structural variation such as insertions, deletions, and inversions
(International Human Genome Sequencing Consortium 2004;
Tuzun et al. 2005; Kidd et al. 2008).
Massively parallel genome-sequencing technologies no lon-
ger rely on cloning DNA fragments in a bacterial host. The plat-
forms currently on the market (454, Illumina, SOLiD, Ion Torrent)
replaced vectors with synthetic adapters and bacterial colonies
with PCR-amplified ‘‘clones’’ of DNA fragments tethered to a bead
(Margulies et al. 2005; McKernan et al. 2009) or with ‘‘colonies’’ of
identical molecules grown by bridge PCR amplification on a solid
surface (Bentley et al. 2008).
However, none of these platforms can handle DNA mole-
cules much longer than 1 kb. Consequently, paired-end se-
quencing of DNA fragments >1 kb by these technologies requires
‘‘jumping’’ constructs (Collins and Weissman 1984; Poustka et al.
1987): the ends of size-selected genomic DNA fragments are
brought together by circularization, the bulk of the intervening
DNA is excised, and the coligated junction fragments are isolated
Suitable protocols exist for converting sheared and size-
selected DNA samples to jumping libraries and for generating read
pairs that span several kb of genomic distance which is generally
sufficient to fashion accurate and highly contiguous de novo assem-
blies of microbial genomes from massively parallel short sequencing
reads (MacCallumet al. 2009;Nelson et al.2010; Nowrousian et al.
2010). However, early short-read assemblies of complex genomes,
including human genomes, turned out fragmented—despite jumps
up to ;12 kb in length (Li et al. 2010a,b; Schuster et al. 2010; Yan
et al. 2011). Without the equivalent of Fosmid or BAC end se-
quences, the N50 scaffold length (a measure of long-range con-
nectivity) of these assemblies was <1.3 Mb. By comparison, largely
owing to paired-end reads from large-insert clones, some of the best
traditional Sanger-based mammalian draft assemblies had N50
scaffold lengths of >40 Mb (Lindblad-Toh et al. 2005; Mikkelsen
et al. 2007).
Constructing a jumping library entails numerous physical and
enzymatic DNA manipulations. Several steps, notably size selection
and circularization of genomic DNA fragments in vitro, become
increasingly difficult and inefficient as the desired jump length,
and hence, fragment length, goes up. In contrast, Fosmid cloning
employs a sophisticated biological machinery to carry out these
critical steps: Large fragments are size-selected (and short com-
petingfragments excluded)by packaging in bacteriophage l; once
inside the Escherichia coli host, cohesive ends mediate efficient
circularization—aided by the cellular machinery and a powerful
selection for circular amplicons.
To our knowledge, no jumping library constructed to date
from uncloned DNA fragments has approached the average span
(35–40 kb) and complexity (>105independent clones per mg of
1Present address: Novartis, Cambridge, Massachusetts 02139, USA.
Article published online before print. Article, supplemental material, and pub-
lication date are at http://www.genome.org/cgi/doi/10.1101/gr.138925.112.
22:2241–2249 ? 2012, Published by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/12; www.genome.org
gap, we and others have taken a hybrid approach wherein Fosmid
libraries are constructed first and then converted to Fosmid-size
jumps in vitro (Gnerre et al. 2011; Hampton et al. 2011).
Here, we present the experimental details of the ‘‘Fosill’’
concept (Gnerre et al. 2011) as well as extensive improvements of
the original protocol. The term Fosill stands for paired-end se-
quencing of Fosmid libraries by Illumina, though we note that this
approach should work for any massively parallel sequencing
technology that can generate paired reads. We describe the meth-
odologyand novelcloning vectors thatenablemolecular barcoding
of DNA inserts and multiplex Fosmid library construction without
physical size selection of sheared genomic DNA. We demonstrate
the power of Fosill to detect structural abnormalities in cancer ge-
nomes and to improve de novo assemblies of mammalian ge-
nomes from short reads.
Modified Fosmid cloning vectors
To facilitate the conversion of Fosmids to Illumina-compatible
Fosill jumping libraries, we modified the original Fosmid vector
pFOS1 (Kim et al. 1992) such that the cloning site is flanked by
Illumina forward and reverse sequencing primers and a pair of
Nb.BbvCI nicking endonuclease sites (Fig.1A). Ourmodified family
of four pFosill vectors retains salient features of pFOS1 such as dual
cos sites (Evans et al. 1989) and a pUC-derived pMB1 origin of rep-
lication which facilitates the preparation oflarge amountsofpFosill
plasmid. In vitro packaging in bacteriophage l removes the pUC
portion of the plasmid, thereby rendering a single-copy amplicon
under the control of the F-factor origin of replication oriS.
Genomic DNA fragments prepared by random shearing, end-
repair, and size selection to ;35–45 kb can be inserted by blunt-
downstream from the sequencing primers (Fig. 1B). Alternatively,
using pFosill-3 and -4, DNA fragments can be ligated first to an
excess of SapI adapters and then inserted by sticky-end ligation
(Fig. 1C). The adapters serve two purposes: First, capping the ends
with non-self-complementary three-base overhangs helps prevent
coligation artifacts and enables Fosmid cloning of sheared geno-
mic DNA without physical size selection; second, genomic frag-
mentscan be taggedwithuniquebarcodesin theadapters,thereby
allowing pooled Fosmid cloning of multiple DNA samples at once.
pFosill vectors are equipped with either SBS-8 (pFosill-1 and -3) or
SBS-12 (pFosill-2 and -4) reverse Illumina primer sequences. Thus,
the system is compatible with single- and multiplex paired-end
digestion makes two symmetrical single-strand nicks, each located
59 of the cloned fragment (Fig. 2A,B). Then, in the presence of
dNTPs, DNA polymerase I translates the nicks in opposite di-
rections into the insert (Fig. 2C). The insert is then fully cleaved at
nickedsites usingS1 nuclease whichreleases allbutthe endsof the
cloned insert from the vector backbone (Fig. 2D). This is analogous
to the nick-translation-directed cleavage protocol used to con-
struct jumping libraries for SOLiD sequencing (McKernan et al.
are being nicked as well and give rise to fragments that are no
longer attached to the vector.
modified pFOS1 Fosmid vectors. The cloning site for inserting the geno-
mic DNA fragments is flanked by forward and reverse Illumina-primer
sequences(ILMN-F andILMN-R)and twoNb.BbvCI nicking endonuclease
sites. Nicks (yellow triangles) are introduced on two different strands and
are located 59 of the cloning site. ILMN-F is the standard Illumina se-
quencing primer SBS-3. The reverse primer in pFosill-1 and pFosill-3 is the
the reverse primer is SBS-12 for three-read multiplex paired-end se-
quencing. The pUC-derived portion between the two cos sites is not
present in the final circularized Fosmids which replicate under the control
of oriS and the F-factor functions repE and sopA-C that ensure proper
partition of the Fosmid among the two daughter cells. Vectors are cut at
the unique AatII site as well as two restriction sites at the cloning site
and dephosphorylated. (B) Cloning site of pFosill-1 (SBS-8 version) and
pFosill-2 (SBS-12). Sheared, end-repaired, and size-selected genomic in-
sert fragments are inserted by blunt-end ligation between two dephos-
phorylated Eco72I sites 4 bp downstream from the ILMN sequencing
primers. The SapI sites shown are not useful for cloning as pFosill-1 and -2
harbor three additional SapIsites.(C) pFosill-3 (SBS-8version) and pFosill-4
(SBS-12) are digested with SapI which excises a single fragment that
includes the 39 ends of the sequencing primers. Sheared and end-repaired
genomic insert fragments are ligated to an excess of adapters that provide
an 8-bp barcode (orange), the 39 end of the Illumina sequencing primers,
and three non-self-complementary 59 overhanging bases for sticky-end
ligation to the SapI ends of the vector arms. Supplemental Table S1
summarizes the relevant features of all four pFosill vectors.
pFosill cloning vectors. (A)GeneralmapofthepFosillfamilyof
Williams et al.
After circularization by intramolecular ligation (Fig. 2E), the
coligated insert termini are flanked by Illumina forward and re-
verse sequencing primer binding sites and can be PCR-amplified
can be controlled to some degree by the
duration of the nick-translation (Supple-
the Fosill library is ready for paired-end
sequencing by Illumina.
Testing and optimizing Fosill library
To test the Fosill strategy, we constructed
a Fosmid library from 30 mg Schizosac-
charomyces pombe genomic DNA. The
library size, as estimated by plating small-
scale test transductions on chloramphen-
units (cfu). We performed a large-scale
transduction with the remainder of the
packaged library, amplified transductants
in bulk by overnight liquid culture at
30°C,andpreparedFosmid DNA.We then
converted 10 mg of Fosmid DNA to a Fosill
jumping library, sequenced it in 2 3 101-base paired-end mode on
a GAII instrument, and aligned the reads to the S. pombe reference
genome sequence (Table 1, library S).
Of 18.1 million unambiguously placed read pairs, 17.1 mil-
lion (94%) had the expected spacing (30–50 kb) and orientation
(convergent). On average, these bona fide correct Fosmid jumps
were 37.8 kb in length with a standard deviation of 3.4 kb. Less
than 1% of the aligned read pairs were obvious chimeric artifacts
that jumped wider than 100 kb or across chromosomes. The chi-
merism rate of the nonredundant set of unique read pairs (1.71
million) was higher (4.7%; Supplemental Table S1), presumably
because singular artifacts and mapping errors have greater weight
in thiscalculation.Atthisdepthofsequencing(;123), werecover
essentially all unique 30- to 50-kb jumps present in the Fosill li-
brary. Accordingly, the total number of unique Fosmid-size jumps
was 1.47 million, approximately the same as the estimated size of
the original Fosmid library (1.4 million cfu).
A fraction (6.3%) of nonredundant read pairs mapped <1 kb
apart (Fig. 3A). Manual inspection suggested that a majority of
these represented ‘‘nonjumps,’’ i.e., single small contiguous ge-
Whatever the exact molecular mechanism, we reasoned that
these undesired side products would tend to be shorter than av-
erage and enriched in the lower half of the broad smear of PCR-
amplified Fosills (Supplemental Fig. S1). We tested this hypothesis
with our second test Fosmid library, ;6.7 million cfu generated
from 60 mg of DNA from K-562, a well-studied human chronic
myelogenous leukemia (CML) cell line (Lozzio and Lozzio 1975).
windows (450–700 bp and 700–900 bp) and sequenced them sepa-
rately. As expected, the lower size cut contained a higher proportion
30- to 50-kb jumps within each fraction was 5.5 million and 3.8
million. Both size cuts combined had a complexity of 6.9 million
correctly spaced unique read pairs (Table 1, libraries H1 and H2).
For our third test organism, mouse (library M), we sought to
eliminate short nonjumps more thoroughly by repurifying the
Fosill jumping library. (A,B) The two Nb.BbvCI sites in the vector are
nicked. (C) The nicks are translated in opposite directions into the cloned
originating at any BbvCI sites within the genomic DNA sequence. (E)
Fragments are circularized by intramolecular ligation. (F) Recircularized
vector molecules serve as templates for inverse PCR with full-length Illu-
mina enrichment primers that include the sequences required for bridge-
amplification and paired-end sequencing of the coligated termini of the
original Fosmid insert on the Illumina flow cell.
Conversion of a Fosmid library to an Illumina-compatible
Summary statistics for four Fosill libraries
Organism S. pombe Human (K-562) Mouse
Fosmids (million cfu)
M H1 H2
13 Prep gel Low rangeHigh range23 Prep gel
Total read pairs (million)
Unambiguously placed pairsa(million)
Uniquecunambiguously placed pairs (million)
Uniqueccorrect jumps (million)
Mean correct jump length 6 s.d. (kb)
Physical genome coverage
Total unique correct jumpsd(million)
37.8 6 3.4
38.6 6 3.8
38.5 6 3.6
38.4 6 3.5
aRead pairs with both reads aligned to a single region in the genome.
bConvergent read pairs that aligned 30–50 kb apart.
cAfter removal of duplicate read pairs (within each Fosill library) with identical start sites of forward and
reverse sequencing reads.
dAfter removal of all duplicate read pairs (for each organism) with identical start sites of forward and
reverse sequencing reads.
Massively parallel end sequencing of Fosmids
size-selected PCR product on a second preparative gel. Only ;1%
of nonredundant read pairs from these doubly size-selected Fosills
aligned <1 kb apart (Fig. 3C); 96% (5.6 million distinct jumps)
spanned 30–50 kb; 2.4% were classified as chimeric. The rate of
chimerism for all unambiguously placed read pairs (18.7 million)
was 1%. A detailed breakdown of the sequencing reads from all
four test libraries is available in Supplemental Table S2.
To fine-tune the protocol, we used barcoded primers for PCR
amplification, thereby tagging different aliquots of a human
and then combined and sequenced as a pool. Narrowing the size
window by raising the lower size cut-off reduced the percentage of
‘‘nonjumps’’ from 1.8% to 0.5% while maintaining the total
number of correct jumps (Supplemental Fig. S2A). We also found
narrow automated size-fractionation on gel cartridges more effec-
tive than manual gel cuts (Supplemental Fig. S2B).
Fosmid library construction is a low-throughput process,
and size selection of large DNA fragments on a pulsed-field gel is
a particularly cumbersome step and often a source of sample loss.
Multiplexing of samples prior to Fosmid library construction would
increase throughput of the process and potentially reduce sample
loss. To test multiplex Fosmid cloning of DNA fragments without
prior size selection, we prepared four aliquots of sheared, end-
and ligated each separately to an excess of barcoded SapI adapters.
We then combined the four tagged reactions, constructed a single,
four-plex barcoded Fosmid library and converted it to Fosills.
Only 0.6% of all read pairs had discordant barcodes at the
beginning of forward and reverse sequencing reads (Supplemental
Table S3), suggesting a low rate of ‘‘recombination’’ between Fos-
mids during the conversion process. It should, therefore, be pos-
sible to construct tagged libraries in multiplex format for several
genomes at once.
mapped read pairs ranged from 4.8 to 8.9 million and represented
0.30 to 0.52 million unique correct jumps per sublibrary (Table 2),
i.e., 0.15 to 0.26 million per mg of input DNA. Mean spacings and
standard deviations of correct jumps were similar to those from
a side-by-side comparison of jump-size distributions). The rate of
pairs) was slightly higher than for the ‘‘traditional’’ mouse Fosill
library (1% and 2.4%; Supplemental Table S2) but still acceptable.
Based on these data, we conclude that multiplexed gel-free Fosmid
hands-on and in a shorter amount of time.
Detection of structural rearrangements
To assess the power of Fosills to detect gross structural rearrange-
ments, we searched for loci in the K-562 genome spanned by jumps
which were aberrantly spaced or oriented or interchromosomal in
the human reference genome. We identified 21 distinct rearrange-
ments with 10 or more independent supporting read pairs (Sup-
observed events is listed in Table 3. While the K-562 is a well-
studied cancer cell line, and many structural abnormalities in its
genome are known, the data analysis was performed in a blind
The t(9;22) translocation that gives rise to the BCR-ABL1 fu-
sion protein was framed by a total of 887 unique read pairs that
appear chimeric when aligned to the reference genome. The large
number of BCR-ABL1 hits is consistent with extensive amplifica-
tion of this locus (Ross et al. 2009). Given the complexity (6.9
million) and average spacing (38.5 kb) of Fosill jumps, one would
expect ;90-fold coverage for a single-copy locus.
We detected two more rearrangements, a tandem duplication
on chromosome 6 and a second t(9;22) translocation, that could
plausibly encode in-frame fusion proteins. Of note, chimeric
transcripts supporting all three gene fusions (BCR-ABL1, BAT3-
SKC44A4 and NUP214-XKR3) have been previously identified in
2010). We were able to pinpointboth the BCR-ABL1 andthe BAT3-
SKC44A4 junctions by 32 and seven ‘‘chimeric’’ single Fosill se-
quencing reads, respectively. The BCR-ABL1 junction matched the
junction sequence reported in the literature (Chissoe et al. 1995;
Shibata et al. 2010). Maps showing the location of breakpoints and
read pairs implicating the three gene fusions and the other two
rearrangements listed in Table 2 (an inversion and a deletion event)
are available in Supplemental Figure S4.
end Fosill sequences. Shown are smoothed histograms of the spacing
between unique read pairs in Fosill libraries from S. pombe 972h (A), hu-
man K-562 library H1 (gray) and H2 (black) (B), and mouse C57BL/6J (C)
in their respective reference genomes. (y-axis) Percentage of all unique
read pairs that fall in the 1-kb bin indicated on the x-axis. The percentages
of unique read pairs spanning <1 kb and 30–50 kb are indicated.
Length distribution of genomic distance spanned by paired-
Williams et al.
2244 Genome Research
Impact on de novo genome assemblies
To demonstrate the effect of Fosill jumps on the long-range con-
nectivity of de novo genome assemblies, we performed three
Illumina-based ALLPATHS-LG draft assemblies (Gnerre et al. 2011)
N50 scaffold length of 2.8 Mb. Adding data from the Fosill library
(80-fold physical coverage) improved the N50 scaffold length to
17.0 Mb, rivaling the long-range connectivity (16.9 Mb) of the
capillary-sequencing-based draft assembly 3 (Waterston et al.
2002). The scaffold accuracy, defined as the percentage of pairs of
loci that were 100 kb apart in the assembly and had matching
spacing and orientation in the reference genome as described
previously (MacCallum et al. 2009; Gnerre et al. 2011), was es-
sentially the same for all three assemblies.
Avoiding the cloning step in a microbial host has been a major
factor in the efficiency and economy of massively parallel se-
quencing technologies. However, relying solely on enzymatic re-
actions in vitro to circularize and amplify genomic DNA fragments
has made it a major technical challenge to produce the modern
equivalent of the Fosmid- or BAC-end sequences that were crucial
for assembling and analyzing complex genomes in the past. Fosill
is a hybrid approach that employs packaging in bacteriophage l
and cloning in E. coli, followed by in vitro manipulations and PCR
amplification to convert Fosmids to Fosmid-sized Illumina jumps.
After library amplification by simple outgrowth in liquid culture,
each Fosmid clone is represented multiple times. Hence, unavoid-
able DNA losses during the subsequent in vitro manipulations do
not necessarily cause a concomitant drop in library complexity.
In this respect, Fosills are similar to Fosmid ‘‘diTags’’ (Hampton
et al. 2011). The principal advantage of our method is that it al-
lows much longer sequencing reads (up to 2 3 101 bases in the
current study, but even longer reads are possible), whereas the
EcoP15I digest strictly limits the diTags to 2 3 26 bases. Fosill reads
have therefore more power to discriminate between different in-
stances of repeat elements or segmental duplications, a significant
benefit for mapping and assembly, particularly of mammalian and
human genomes. We optimized Fosill using genomic DNA from
three organisms(fission yeast, mouse,and
human) and carried out pilot studies to
identification and mapping of chromo-
somal rearrangements and de novo as-
sembly of complex genomes.
In the first pilot study, we found 21
gross abnormalities in the well-studied
CML cell line (K-562), each event sup-
ported by at least 10 unique jumps. Three
of them give rise to gene fusions that are
corroborated by RNA-seq data. Scanning
a genome with Fosill read pairs requires
fewer read pairs than with short-range
jumps or by direct paired-end sequencing
of fragments. For example, 7.4 million
unique Fosill jumps contained 887 read
pairs implicating the hallmark t(9;22)
translocation in the K-562 genome. This
structural variant can be detected by se-
quencing a standard ;300-bp fragment
library. However, the detection sensitivity is ;400-fold lower (175
implicating hits in 611 million unique read pairs; C-Z Zhang and
M Meyerson, pers. comm.). If we count all aligned read pairs, in-
cluding duplicates, (43.6 million Fosill jumps vs. 685 million stan-
dard read pairs), Fosill sequencing is 80 times more sensitive.
Perhaps more importantly, considering the low cost of sequencing,
long-distance jumps can span breakpoints that are flanked by long
stretches of repetitive sequence on either side. They are also more
the reference genome than expected for bona fide Fosmid ends
(International Human Genome Sequencing Consortium 2004).
The impact of Fosill on de novo assemblies of the mouse
genome was profound. It boosted the N50 scaffold length from
2.8 Mb to 17.0 Mb without compromising the scaffolding accu-
racy. By this measure, to our knowledge, our Fosill-powered assem-
bly has better long-range connectivity than any Fosill-free de novo
short-read assembly of a mammalian genome reported to date.
For these studies, we sequenced deeply and generated re-
dundant read pairs to maximize the yield of unique Fosill jumps.
This adds relatively little cost to a genome project, for the total
numberof Fosillreads is smallcomparedto the requirednumberof
short-insert reads. In almost all cases, the yield of distinct jumps of
the expected length corresponded well to the estimated size of the
initial Fosmid library, indicating little, if any, loss of complexity
during the conversion process.
Our current set of modified Fosmid vectors enables barcoding
and multiplexing at two stages. Barcodes can be introduced during
Barcoded Fosill jumps from a multiplex Fosmid library
Barcode (read 1/read 2)A/AB/BC/C D/D
Unambiguously placed pairsa(million)
Chimeric read pairsc(%)
Uniquedunambiguously placed pairs (million)
Unique correct jumps (million)
Chimeric uniquely placed read pairsc(%)
Mean correct jump length 6 s.d. (kb) 37.8 6 3.637.5 6 3.337.8 6 3.6 37.8 6 3.6
Fosill libraries prepared from sheared mouse DNA using barcoded SapI adapters and no size-selection
before ligation to the cloning vector.
aRead pairs with both reads aligned to a single location in the mouse genome after trimming the
barcodes at the beginning of each read.
bConvergent read pairs that aligned 30–50 kb apart.
cUnambiguously placed read pairs aligned either in the wrong orientation, >100 kb apart, or to different
contigs of the reference mouse genome sequence.
dAfter removal of duplicate read pairs (within each barcoded sublibrary) with identical start sites of
forward and reverse sequencing reads.
identified by Fosill
Examples of rearrangements in the K-562 genome
aUnique read pairs.
bRanked by number of supporting unique read pairs.
Massively parallel end sequencing of Fosmids
the final PCR amplification of the Fosill library for standard three-
read multiplex paired-end sequencing. ‘‘Inline’’ barcodes can be
added by ligating excess amounts of barcoded SapI adapters to tag
genomic DNA fragments before ligating them to the Fosmid
cloning vector. These adapters also help prevent coligation of un-
related DNA fragments and thus the formation of packageable
a preparative gel or sucrose gradient, i.e., relying exclusively on
bacteriophage l for size selection. Based on our experience so far,
gel-free Fosmid cloning streamlines the process significantly, in-
produces Fosill libraries of acceptable size and quality.
We expect multiplex Fosmid-library construction from mul-
tiple DNA samples at once to be most useful for smaller genomes.
There are also less obvious benefits of pooling multiple differently
tagged aliquots of the same DNA sample, particularly for large ge-
read improves the optical separation of adjacent clusters on the
Illumina flowcell and thus allows higher read densities; second,
filtering out read pairs with discordant barcodes may remove chi-
meric artifacts that arose downstream from Fosmid cloning.
Despite these improvements, Fosmid cloning remains low-
throughput and sensitive to the quantity and quality of the input
DNA. Fosmid libraries are also subject to cloning bias. While Fosill
appears to work for the extremely GC-rich (69%) genome of Rho-
dobacter sphaeroides (not shown), we expect cloning problems for
extremely AT-rich DNA from organisms such as Dictyostelium dis-
coideum or Plasmodium falciparum that proved recalcitrant to clon-
ing as Fosmids or BACs in the past (Gardner et al. 2002; Eichinger
comprehensive to support successful and high-profile genome
projects (Osoegawa et al. 2000, 2001; Wei et al. 2007) and that
sequencing libraries constructed without cloning have biases of
In principle, one can easily modify other cloning vectors, for
example, plasmid or BAC vectors, and generate shorter ‘‘Plasill’’ or
longer ‘‘BACill’’ jumps. The former may or may not be a practical
alternative to standard 3–10 kb in vitro jumping libraries. The
latter would extend the jump range up to ;200 kb. In routine
practice, considering the steep drop in cloning efficiency for DNA
realistic proposition. We note, however, that BACs have a much
wider size distribution than Fosmids. Thus, in our view, Fosmid
jumps currently occupy the sweet spot, the best balance of cloning
efficiency and jump range. Not only do they help assemble ge-
nomes, their tight size distribution also allows genome-wide scans
of individuals by consistently spaced read pairs to analyze struc-
tural polymorphisms in the human population and to map gross
rearrangements that cause or contribute to human disease.
Construction and preparation of pFosill cloning vectors
pFosill-1 was constructed by inserting a cloned synthetic DNA
(New England Biolabs [NEB]). Replacing pFosill-1 sequences be-
tween the BamHI site and SfiI sites with Illumina SBS-12 primer
sequences and an Nb.BbvCI nicking site resulted in pFosill-2.
pFosill-3 is a derivative of pFosill-1 that has all SapI sites (and
several other restriction sites) outside of the Illumina primer se-
quences removed. pFosill-4 is a derivative of pFosill-3 containing
Illumina SBS-12 instead of SBS-8 primer sequences.
pFosill plamids were propagated in E. coli Stbl2 cells (Invi-
trogen) grown at 30°C in LB or TB broth containing 100 mg/mL
carbenicillin and 15 mg/mL chloramphenicol. A 200-mg aliquot
of a Qiafilter plasmid mega preparation (Qiagen) was incubated
for 30 min at 37°C in 500 ml containing 300 units ‘‘plasmid-safe’’
E. coli chromosomal DNA fragments. After heat inactivation
(30 min at 70°C), the reaction was cleaned up with a 1.8-fold vol-
ume of AMPure XP beads (Beckman Coulter Genomics). The beads
were washed according to the manufacturer’s protocol and the
plasmid DNA eluted in 200 ml T10E0.1buffer.
Vectors were prepared for cloning as follows, using restriction
endonucleases from Fermentas. pFosill plasmid (50 mg) was di-
gested with 200 units AatII and either 200 units Eco72I (pFosill-1
and pFosill-2) or 200 units LguI, a SapI isoschizomer (pFosill-3 and
pFosill-4). After 1 h at 37°C and heat inactivation for 20 min at
65°C, the reaction was cleaned up with a 1.8-fold volume of
AMPure XP beads. The restriction fragments were eluted in 200 ml
and 1 h at 55°C) with 2 3 25 units calf intestine alkaline phos-
phatase (NEB). The vector arms were cleaned up by two successive
extractions with phenol/chloroform/isoamylalcohol, precipitated
with ethanol, and resuspended in T10E0.1to a final concentration
(f.c.) of 0.5 mg/ul.
Preparation of genomic DNA fragments
S. pombe strain 972h was a kind gift of Nick Rhind (U. Mass.
Medical School). K-562 cells were kindly provided by Robyn Issner
(Epigenomics Program, Broad Institute). DNA from Mus musculus
strain C57BL/6J was from the Jackson Laboratory. DNA from
a normal humanlymphoblastoidcellline(NA12892)was fromthe
Coriell Institute. The preparation of genomic DNA fragments for
Fosmid cloning was a modification of established protocols.
Briefly, genomic DNA (typically two 15-mg aliquots in 125 ml
T10E0.1) was HydroSheared (Digilab) by 60 passages at speed code
15 through a 0.0060 shearing assembly (Bird Precision). The frag-
ments were end-repaired for 30 min at 20°C in 175 ml containing
13 T4 DNA ligase buffer, 0.25 mM dNTPs, 15 units T4 DNA
assemblies of the mouse genome
Long-range connectivity of three de novo draft
Physical coverage by XL jumpsd
N50 scaffold length (Mb)e
aAssembly based on paired-end reads from ;180-bp fragment libraries
and jumping constructs spanning up to 10 kb.
bLiterature data (Waterston et al. 2002).
cExtra long read pairs generated directly or indirectly from Fosmid or BAC
dNonredundant set of unique jumps.
eUngapped scaffold lengths, i.e., total length of contigs within each
fPercentage of randomly chosen pairs of loci that spanned 100 kb in the
assembly that had essentially the same spacing and orientation in the
Williams et al.
2246 Genome Research
to a f.c. of 50 mM and heat inactivation for 10 min at 70°C.
For Fosmid cloning of size-selected DNA, 7.5 mg end-repaired
fragments were loaded into a 42-mm-wide well on a 1% SeaPlaque
GTG (Lonza) 0.53 TBE agarose gel and run at 14°C on a CHEF-
DRIII system (BioRad) set to 6 V/cm, 120°, and a switching time
ramped from 1.2 to 6 sec over 19 h, along with 8.3 kb to 48.5 kb
DNA size markers (BioRad). Marker lanes were cut from the gel and
a Dark Reader Transilluminator (Clare Chemicals) and a gel slice
between the positions of the 33.5 kb and 48.5 kb size markers ex-
cised from the unstained preparative portion of the gel. The gel
slice was equilibrated twice against two volumes of 13 b-agarase
buffer (NEB) supplemented with 40 mM NaCl for 30 min each on
After cooling to 42°C, the agarose was digested at 42°C by two
successive 2-h incubations using 1/100th gel volume of 1 unit/ml
b-agarase (NEB) each. After heat inactivation at 70°C for 10 min,
the tube was chilled on ice for 5 min and centrifuged at 4°C for 20
min at 10,000 rpm. The volume of the supernatant (;2 mL) was
reduced to ;350 ml by centrifugal ultrafiltration at 2000g using an
Amicon Ultra, 0.5-mL 100K concentrator (Millipore). The size-se-
lected genomic DNA was cleaned up by two successive extractions
with phenol/chloroform/isoamylalcohol, ethanol precipitated,
and resuspended overnight in 20 ml T10E0.1.
For multiplex Fosmid cloning without size selection, bar-
coded SapI adapters were annealed from oligonucleotide pairs 59-
[PHOS]GATCTXXXXXXXX and 59-[PHOS]XXXXXXXXAG, where
fragments were heated at 70°C for 10 min, spun on Amicon Ultra
0.5-mL 100K centrifugal concentrators, and cleaned up by three
washes with 500 ml T10E0.1(;10 min at 2000g for each step).
Concentrated fragments (;30 ml) were ligated to 115 ng of pre-
annealed barcoded SapI adapter at 16°C for 2 h in 100 ml con-
taining 13 T4 DNA ligase buffer (NEB) and 1000 units T4 DNA
ligase (NEB). The ligations were inactivated at 70°C for 10 min,
pooled, cleaned up, and concentrated to ;30 ml on Amicon Ultra
0.5-mL 100K centrifugal concentrators spun at 2000g as described
Fosmid library construction
and pFosill-4 for cloning non-size-selected, SapI-adapter-ligated
total amount of insert DNA fragments. Each 10-ml ligation con-
tained 250 ng inserts, 500 ng cut and dephosphorylated pFosill
vector, 13 T4 DNA ligase buffer, and 2000 units T4 DNA ligase
(NEB), and was incubated overnight at 25°C. The ligations were
heat-inactivated at 70°C for 10 min, split into 2 3 5 ml, and
packaged using MaxPlax l packaging extracts (Epicentre). Each 5-ml
aliquot was packaged by two successive additions of 25 ml pack-
aging extract and 90-min incubations at 30°C. After adding 940 ml
Phage dilution buffer (SM buffer with 0.01% gelatin) and 70 ml
DMSO (Sigma-Aldrich), packaged Fosmid libraries were titered and
stored at ?80°C.
Batches of l-competent T1-resistant E. coli GC10T1 cells,
frozen and stored at OD600;0.1, were prepared by standard pro-
tocols (Garnes et al. 2003). Thawed cells (1 mL) were mixed with
9 mL 10 mM MgSO4and 1 mL packaged Fosmid library. After 20
min at room temperature, 40 mL of prewarmed (37°C) LB broth
was added and the incubation continued for 45 min. at 37°C with
shaking at 225 rpm. The number of Fosmid clones was estimated
by plating 40 ml of the 51-mL culture onto LB agar plates supple-
mented with 15 mg/mL chloramphenicol. Three 51-mL cultures
were combined into a 2-L flask containing 600 mL 23 YT media
supplemented with 15 mg/mL chloramphenicol and grown over-
night at 30°C with shaking. Fosmid DNA was isolated from the
Conversion of Fosmids into Fosills
Ten mg of Fosmid DNA were nicked for 1 h at 37°C in 250 ml
containing 13 NEB Buffer 2 (NEB), 13 BSA (NEB), and 50 units
Nb.BbvCI (NEB). Products were cleaned up using 1.8-fold volume
AMPure XP beads (Beckman Coulter). Nicked Fosmids (800 ng)
(NEB), 0.25 mM dNTP, and 50 units DNA polymerase I (NEB). The
nick translation was stopped by adding EDTA to a f.c. of 50 mM.
Productswerecleanedup using 1.8-foldvolumeAMPureXP beads.
Nicks were cleaved at 37°C for 15 min in 50 ml containing 300 mM
NaCl, 13 S1 buffer, and 200 units nuclease S1 (Invitrogen). The
reaction was stopped by EDTA (50 mM f.c.) and cleaned up using
1.8-fold volume AMPure XP beads. Ends were repaired at 20°C for
30 min in 100 ml containing 13 T4 Ligase Buffer, 0.25 mM dNTP,
9 units T4 DNA polymerase, 30 units T4 PNK, and 5 units Klenow
DNA polymerase (all from NEB). After adding EDTA to 50 mM f.c.,
DNA was cleaned up using 1.03 AMPure beads and recircularized
overnight at 16°C in 500 ml containing 13 T4 Ligase buffer and
4000 units T4 ligase (NEB). Products were purified using a Qiagen
PCR clean-up kit and eluted in 50 ml T10E0.1. To determine the
test PCR reactions were set up containing 13 Phusion HF Master-
mix (NEB), 2 ml of circularized DNA, and 0.5 mM PCR primers:
PE1.0 andPE2.0 primers(Illumina) for single-plex Illuminapaired-
3); 59- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTA
CACGACGC and 59- CAAGCAGAAGACGGCATACGAGATXXXX
XXXXGTGACTGGAGTTCAGACGTGTGC for three-read multi-
plex paired-end sequencing runs with X8denoting the eight-base
PCR reactions were split into four 10-ml aliquots and run on
GeneAmp 9700 thermocyclers (Applied Biosystems) as follows:
98°C for 30 sec; 12, 15, 18, or 21 cycles of 98°C for 10 sec, 65°C for
30 sec, and72°Cfor30 sec; anda finalextensionfor7 min at 72°C.
PCR products were run on Criterion 5% 13 TBE polyacrylamide
gels (Bio-Rad) and stained with SYBR Green I. The optimal number
N of PCR cycles determined from this gel was used for amplifica-
tion of the remaining library in a 600-ml PCR reaction containing
48 ml circularized DNA, 13 Phusion HF Mastermix (NEB), and
0.25 mM of appropriate PCR primers. Thermocycling in 50-ml ali-
quots was as follows: 98°C for 3 min; N cycles of 98°C for 120 sec,
65°C for 30 sec, and 72°C for 30 sec; and a final extension at 72°C
for 7 min. The PCR product was purified using 1.8-fold volume
AMPureXP beadsandeluted in 30 ml T10E0.1. ThePCR productwas
an automated Pippin 1.5% agarose DNA gel (Sage Science) with
size-selection settings of 550–900 bp. The latter resulted in a 550-
to 800-bp Fosill PCR product. PCR products size-selected by Pippin
were cleaned up using 1.8-fold volume AMPure XP beads and
sequencing chemistry on Illumina GAII or HiSeq instruments.
Paired 76-base or 101-base Illumina reads were aligned to the
S. pombe 972h (NC_003424.2, NC_003423.2, NC_003421.2), Mus
Massively parallel end sequencing of Fosmids
musculus C57BL/6J (NCBI37/mm9), or Homo sapiens (GRCh37/
hg19) reference genome sequences by BWA v0.5.9 (Li and Durbin
2009). Each read was aligned independently with bwa aln (-q 5 -l
32 -k 2 -t 4 -o 1), and then the paired alignments were combined
using bwa sampe (-a 100000). MergeBamAlignments, from the
picard package (http://picard.sourceforge.net/) v1.59, was used to
return the unmapped reads to the aligned BAM file. A custom
definitions: (1) unambiguously mapped read pairs: pairs with both
(2) duplicate read pairs: pairs where both reads have identical start
sites of forward and reverse sequencing reads; (3) correct jumps:
read pairswherethe readsfaceeach other andare aligned 30–50kb
apart; (4) chimeric jumps: (a) pairs with unexpected orientation
aligning to the same strand in the same orientation), and (b) pairs
with unexpected spacing (>100 kb or aligning to different contigs
in the reference genome sequence, usually different chromo-
somes). ‘‘Inline’’ barcodes in Fosill read pairs derived from SapI
cloning adapters were identified and quantified by comparing to
the first eight bases of each read and requiring a perfect match to
the expected barcode sequence. Chromosomal rearrangements
were identified by dRanger (MS Lawrence, Y Drier, C Stewart, SB
Gabriel, ES Lander, and G Getz, in prep.) as read pairs that map to
different chromosomes or with unexpected spacing (>50 kb) or
orientation. Single reads aligning to either side of a dRanger
breakpoint were identified by BreakPointer (Y Drier, MS Lawrence,
SL Carter, C Stewart, SB Gabriel, ES Lander, M Meyerson, R
Beroukhim, and G Getz, in prep.). A high-level description of
these tools can be found in Berger et al. (2011).
Cloning vectors described in this work are available to academic
GenBank (http://www.ncbi.nlm.nih.gov/genbank) under accession
numbers JX069761 (pFosill-1), JX069762 (pFosill-2), JX069763
(pFosill-3), and JX069764 (pFosill-4). Illumina sequencing reads
have been submitted to the NCBI Sequence Read Archive (SRA)
(http://www.ncbi.nlm.nih.gov/sra) under BioProject ID 40079,
accession number SRX116276 for library S, ID 51977 (SRX029163,
SRX029164) for library M, ID 82383 (SRX118400 and SRX118399)
NA12892 libraries (SRX118354, SRX118355, and SRX118352
for 450, 500, and 550 lower size cut-offs; SRX116629, SRX116621,
and SRX116628 for 23 gel, 13 gel, and Pippin), and ID 51977
LG mouse genome assembly 2 has been deposited in GenBank
under accession number AEKQ02000000 and is available at ftp://
We thank the staff of the Broad Institute Sequencing Platform for
generating sequencing data, Jim Bochicchio and Carsten Russ for
catalyzing project and data submissions, Nick Rhind for S. pombe
972h, Robyn Issner for K-562 cells, and Oleg Iartchuk who sug-
gested converting Fosmid libraries to Illumina jumping libraries
more thanfive yearsago. Thisproject has beenfunded in partwith
federal funds from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Department of Health and
Human Services, under Contract No. HHSN272200900018C, and
with funds from the National Human Genome Research Institute
(HG003067-05 through HG003067-10).
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and A.G. coordinated and directed the project, and L.J.S.W. and
A.G. wrote the paper. L.J.S.W. and A.G. are listed as inventors on
a related patent application filed by the Broad Institute.
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Received February 9, 2012; accepted in revised form June 25, 2012.
Massively parallel end sequencing of Fosmids