De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth

Michael A Fisher
Kara L Mckinley
Luke H Bradley ¤c
Sara R Viola ¤d
Michael H Hecht firstname.lastname@example.com¤a

Departments of Chemistry and Molecular Biology
Department of Biology
Princeton University
PrincetonNew JerseyUnited States of America

Departments of Anatomy and Neurobiology, Molecular and Cellular Biochemistry
Massachusetts Institute of Technology
CambridgeMassachusetts

¤d Current address: Columbia University Medical Center
University of Kentucky
Lexington, New YorkKentucky, New YorkUnited States of America, United States of America

De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth

Coli and Enable Cell Growth. PLoS ONE
6115364Editor: Mark Isalan, Center for Genomic Regulation, Spain Received August 26, 2010; Accepted November 11, 2010; Published January 4, 2011Citation: Fisher MA, McKinley KL, Bradley LH, Viola SR, Hecht MH (2011) De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Current address: Energy Biosciences Institute, University of California, Berkeley, California, United States of America ¤b Current address: United States of America ¤c Current address:
A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of .10 6 de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains -DserB, DgltA, DilvA, and Dfes -were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by coexpression of four novel sequences. Thus, cells deleted for ,0.1% of the E. coli genome (and ,1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo.

Introduction

In 1906 Jacques Loeb suggested that the synthesis of life is a significant goal of biology [1]. A century later, the construction of natural genomes from off-the-shelf chemicals [2,3] demonstrates significant progress toward achieving this goal. Until now, however, most advances in synthetic biology have relied on collections of parts -genes, proteins, and regulatory elementsderived from sequences that already exist in nature. Must the toolkit of life be so restricted? Natural sequences comprise only a miniscule fraction of the theoretical sequence space that is possible for genes and proteins. Indeed, a collection containing just a single molecule of every one of the 20 100 possible 100-residue proteins would fill a volume larger than a mole of universes [4]. From this enormous potential for diversity, natural selection has operated over billions of years to yield a relatively small collection of sequences: Life is sustained by only ,4,000 genes in E. coli and approximately 5-fold more in humans [5,6]. These considerations might lead to the supposition that genes and proteins capable of sustaining life are somehow ''special.'' Is this true? Or might we find functional molecular parts in a collection of artificial sequences designed ''from scratch'' in the laboratory?

To address these questions, we probed the ability of unevolved sequence space to encode functions that enable cell growth. We designed and constructed a collection of artificial genes encoding approximately 1.5610 6 novel amino acid sequences. Because folding into a stable 3-dimensional structure is a prerequisite for most biological functions, we did not construct this collection of proteins from random sequences. Instead, we used the binary code strategy for protein design, shown previously to facilitate the production of large combinatorial libraries of folded proteins [7,8]. In brief, the binary code strategy posits that stably folded proteins can be encoded by specifying the sequence pattern of polar and nonpolar residues (the binary pattern) to coincide with the exposed and buried parts of a structure, respectively. For example, to design a helical bundle comprising a-helices with hydrophilic faces exposed to aqueous solvent and hydrophobic faces buried in the interior of a protein, the binary pattern of polar (P) and nonpolar (N) residues should be PNPPNNPPNPPNNP, consistent with the structural repeat of 3.6 residues per alpha-helical turn [9,10]. For the current studies, we used the binary code strategy to design and construct a library of sequences designed to fold into 102-residue 4-helix bundles. Our strategies for protein design and selecting biological function are summarized in Figure 1. Details describing the construction and biophysical characterization of the library are presented elsewhere [11,12].

Here we demonstrate that novel proteins from this binary patterned library can rescue E. coli cells that lack certain natural proteins required for cell viability. The novel proteins are substantially less active, and may function by different mechanisms than the natural proteins they replace. Nonetheless, co-expression of several novel proteins rescues a strain in which multiple natural genes were deleted simultaneously. These findings show that an organism, which would otherwise not grow, can be sustained by macromolecules devised in the laboratory.

Results and Discussion

De novo designed sequences rescue single-gene knockout strains of E. coli Previous work in our laboratory showed that several purified proteins from our binary patterned alpha-helical libraries can bind a cofactor and perform enzyme-like functions, including peroxidase, lipase, and esterase activities [13]. These findings led us to question whether proteins from this library might also provide biological functions that enable cell growth. To address this question, we tested the ability of binary patterned proteins to rescue strains of E. coli in which a conditionally essential function had been deleted. These strains were obtained from the Keio collection, which contains all the viable single-gene knockout strains of E. coli [5]. We tested 27 auxotrophs that grow on rich media, but fail to grow on minimal media (see legend for Table 1). Each auxotroph was transformed with a library of synthetic genes carried on the expression vector pCA24NMAF2 [14]. Typically, we obtained 5-10 million transformants, thereby ensuring reasonably deep coverage of the library of 1.5610 6 de novo sequences. As negative controls, each auxotroph was transformed with the empty vector or the same vector expressing betagalactosidase. Transformed cells were spread either on LB (rich) or on M9-glucose (minimal) media supplemented with isopropylbeta-D-thiogalactoside (IPTG) to induce expression, and the formation of colonies was monitored (Figures 1 & 2).

On rich media, all transformed cells grew, regardless of whether they received the control lacZ gene or a gene encoding a novel protein. As expected, on minimal media, auxotrophs transformed with the control plasmids failed to grow. (If the negative controls grew, the strain was considered 'leaky' and not used for further studies.) In most cases, auxotrophs transformed with the collection of novel sequences also failed to produce colonies, even after weeks of incubation. This indicates that proteins from the collection could not rescue these strains. However, for four auxotrophs -DserB, DgltA, DilvA, and Dfescolonies formed on minimal plates after several days of incubation (Figures 2A & 2B). Growth of these cells under selective conditions suggests that a novel gene carried by the plasmid complements the deletion. The rates of complementation and the times required for colony formation are summarized in Table 1.

To confirm that the colonies observed on selective media resulted from the uptake of a novel gene and not from an adaptive mutation on the chromosome, colonies were isolated by restreaking, and plasmid DNA was purified; this DNA was then used to transform naïve cells of the same auxotroph, and the transformed cells were once again spread on both LB and minimal plates. Growth of approximately the same number of colonies on selective plates as on rich plates indicates that the observed phenotype (growth on minimal) transferred with the genotype (the plasmid carrying the novel gene). These results are shown in Figure 2C and Figure S1. To ensure that rescue was due to a de novo protein, and not some natural sequence that might have been picked up inadvertently during plasmid constructions, we isolated the ,300 base pair fragment encoding the novel protein, recloned it into a new vector, and showed that the new clone also rescued the deletion strain.

The binary patterned sequences that rescue the four auxotrophs are shown in Figure 3. (Additional sequences are shown in Figure S2.) The novel sequences are designated according to the Binary pattern of polar (red) and nonpolar (yellow) residues designed to fold into 4-helix bundles (7,8). Library construction is facilitated by the organization of the genetic code: Six polar residues (Lys, His, Glu, Gln, Asp, and Asn) are encoded by the degenerate codon VAN, and five nonpolar residues (Met, Leu, Ile, Val, and Phe) are encoded by the degenerate codon NTN. Combinatorial turn positions are encoded by the degenerate codon VRS, which encodes Gln, Glu, Asn, Asp, His, Lys, Arg, Ser, and Gly. (V = A, G, or C; R = A or G; N = A, G, C, or T). Circles with letters indicate fixed residues, and empty circles indicate combinatorially diverse positions. The theoretical diversity of this library is 5 22 66 34 68 12 = 5610 52 . The substantially smaller experimental library (1.5610 6 synthetic genes cloned on a high expression plasmid) was transformed into a strain of E. coli in which an endogenous gene had been deleted (green X). Colony formation on minimal media indicates a novel sequence (purple) enables cell growth under selective conditions. doi:10.1371/journal.pone.0015364.g001 auxotroph they rescue, followed by a number (e.g. Syn-Fes-1 is a synthetic sequence that rescues Dfes.) For the DgltA strain, we isolated one novel protein that enabled cell growth. However, in the other cases, several sequences were isolated, suggesting that deletions of these genes are relatively easy to rescue. As shown by the color-coding in Figure 3, the sequences of the biologically active proteins conform to our binary pattern for the design of 4helix bundles. (As is often true for sequences in combinatorial libraries, spurious mutations were observed occasionally, but these do not interfere with the overall binary pattern -see Figure S2). NCBI-BLAST searches indicate the sequences of the de novo proteins are unlike those of any known natural proteins [15].

The ability of each de novo protein to sustain cell growth was measured in cultures grown in minimal M9-glucose liquid media. Growth rates were compared for deletion strains expressing a de novo protein versus the same strains expressing either the negative control (LacZ) or the corresponding natural protein on the same pCA24NMAF2 vector. As shown in Figure 4, the novel proteins enable cell growth under conditions where auxotrophs transformed with the control plasmid fail to grow at all. Cells relying on the de novo proteins grow significantly slower than those expressing the natural protein. (Fes is an exception because overexpression of E. coli Fes is toxic [14, Genobase ORF JW0576 -http://ecoli. naist.jp/GB6/info.jsp?id = JW0576].) It is not surprising that the unevolved novel sequences function at substantially lower levels than natural sequences selected by billions of years of evolution. Indeed, the relatively slow rates of cell growth enabled by these first-generation de novo sequences suggest that selections for faster growth might facilitate the evolution of more active proteins. Such experiments will provide an opportunity to test whether de novo progenitor sequences might lead to novel evolutionary trajectories.

The results shown in Figures 2 and 4 demonstrate that the de novo sequences enable cell growth; however, viability per se does not indicate that the novel proteins provide the same biochemical activities as the deleted natural proteins. Therefore, we devised a series of experiments to probe the functions of the de novo proteins.

Biological functions of the de novo proteins

The natural genes deleted in these four auxotrophs encode a range of functions (9). These are summarized below and depicted graphically in Figure S3.

N serB encodes phosphoserine phosphatase, responsible for the final step in serine biosynthesis: phosphoserine R serine + phosphate.

N gltA encodes citrate synthase, which catalyzes an early step in glutamate biosynthesis:
oxaloacetate + acetyl-coA R citrate R cis-aconitate R isocitrate R alpha-ketoglutarate R glutamate.
N ilvA encodes biosynthetic threonine deaminase, which catalyzes the first step in the production of isoleucine from threonine:
threonine R 2-ketobutyrate + ammonia R R R isoleucine.
N fes encodes enterobactin esterase, which cleaves the iron-bound enterobactin siderophore, thereby enabling cells to acquire iron in iron-limited environments [16].

In principle, the novel proteins could rescue these auxotrophs either by providing the same function as the deleted enzyme, or by one of the three alternative mechanisms described below:

First, we considered the possibility that an auxotroph in a biosynthetic pathway might be rescued by a de novo sequence that produces the end product via a novel bypass pathway. Although it seems unlikely that small, unevolved proteins could catalyze  novel biosynthetic pathways, we nonetheless tested this possibility. If the novel sequences that rescue DserB, DgltA or DilvA encode bypass pathways for the synthesis of serine, glutamate, and isoleucine, respectively, then these de novo sequences would also rescue cells deleted for enzymes that function at other steps in the these biosynthetic pathways. (Pathways are shown in Figure S3). This possibility was tested by the following three experiments:  (a) De novo sequences that rescued DserB were transformed into DserC cells, which are deleted for phosphoserine aminotransferase. This enzyme converts phosphohydroxypyruvate to phosphoserine in the step prior to that catalyzed by the serB encoded enzyme. (Because the serC gene product is also involved in the biosynthesis of pyridoxine [9], the selective media for this experiment was supplemented with pyridoxine.) (b) The sequence that rescued DgltA was transformed into Dicd cells. This strain is also a glutamate auxotroph because it cannot catalyze the conversion of isocitrate to alphaketoglutarate, the direct precursor of glutamate. (c) The sequences that rescued DilvA were transformed into DilvC and DilvD cells, which are deleted for isomeroreductase and dihydroxyacid dehydratase, respectively. Both these enzymes function downstream of ilvA in the biosynthesis of isoleucine. (Because the ilvC and ilvD gene products are also involved in valine biosynthesis [9], the selective media for these experiments was supplemented with valine.)

In all cases, the de novo sequences did not rescue E. coli cells deleted for functions at other steps in the biosynthetic pathways. This demonstrates that the novel proteins do not enable bypass pathways for the synthesis of their respective end products.

Second, we considered the possibility that our de novo sequences might rescue the auxotrophs by altering the expression or activity of an endogenous E. coli protein. To assess this possibility, we relied on an exhaustive screen by Patrick et al., who identified natural genes whose overexpression can rescue the deletion of noncognate genes in E. coli (i.e. 'multicopy suppressors') [17]. By screening the complete set of overexpressed E. coli ORFs, they found the following multicopy suppressors of our four strains: DilvA can be rescued by overexpression of tdcB [18], or emrD; DserB can be rescued by overexpression of gph, hisB, or ytjC; and Dfes can be rescued by overexpression of thiL or setB. DgltA was not rescued by overexpression of any E. coli genes. To probe whether our novel sequences rely on these E. coli proteins for rescue, we tested whether our proteins rescue the following double deletion strains: DilvADtdcB, DilvADemrD, DserBDgph, DserBDytjC, and DfesDsetB. Transformation with the appropriate novel sequences showed that all five of these double knockouts were rescued by all of the corresponding artificial sequences listed in Figure 3. The ability of our novel sequences to rescue these double deletions shows that the artificial proteins do not function by altering the expression or activity of E. coli proteins known previously to enable rescue.

The double mutant DfesDthiL cannot be constructed because deletion of thiL is lethal. The DserBDhisB strain was constructed, but since DhisB is itself an auxotroph, our novel sequences did not rescue this strain (successful rescue would require the unlikely occurrence of a single de novo protein replacing the activities of two conditionally essential genes.) Therefore, dependence on thiL and hisB could not be assessed. In addition, it remains possible that some other E. coli protein, which escaped selection by Patrick et al., could act as a partner in the rescue mediated by our de novo sequences. With the exception of these caveats, our results demonstrate that the known multicopy suppressors are not required for the biological activities of our de novo proteins.

Third, we considered the possibility that the de novo proteins might rescue the deletion strains by a mechanism that does not depend on the specific sequences (Figure 3), but instead involves global alterations in metabolism that are induced by the mere expression of foreign genes. For example, although our proteins were designed to fold into alpha-helical bundles, we considered the possibility that sequences isolated by our selections might be unfolded, and thereby induce a cellular stress response. To assess folding, we purified several proteins and measured their circular dichroism spectra. These spectra (shown in Figure S4) demonstrate the structures are predominantly alpha-helical, and similar to the spectra of designed 4-helix bundles solved previously by NMR [19,20]. Thus, rescue is not caused by unfolded sequences inducing a stress response.

We also note that if a generic stress response were responsible for rescue, one would expect all the Syn proteins to rescue all the deletions. This is not the case. Syn-Fes does not rescue DgltA, Syn-SerB does not rescue DilvA, and so on. Thus, specific de novo sequences mediate the rescue of specific chromosomal deletions.

To demonstrate explicitly that rescue depends on a specific sequence, rather than a generic cellular response to the expression of foreign genes, we mutated one of the de novo proteins. The design of this mutant was based on an analysis of the common features among the de novo sequences that rescued DilvA. Alignment of the Syn-IlvA sequences revealed two conserved polar residues: Glu36 and Lys42. We constructed the Lys42»Ala mutation in Syn-IlvA-1, and found that although this protein was expressed at the same level as the parental sequence, it fails to rescue DilvA. (These results are shown in Figure S5) Thus, rescue is not simply due to expression of an exogenous gene; it is mediated by sequencespecific features of Syn-IlvA-1.

Fes differs from the other deletions because it is not involved in a biosynthetic pathway. Fes functions in iron acquisition. E. coli secretes the enterobactin siderophore (MW, 670 Da) into the media, where it binds iron, and is transported back into the cell. Because the affinity of enterobactin for iron is extremely high (K D <10 235 M), release of the metal requires degradation of the siderophore. This is catalyzed by the Fes protein, enterobactin esterase [16]. The impact of the de novo syn-fes sequences on iron acquisition is dramatic: Elemental analysis shows that cells expressing the Syn-Fes proteins accumulate 6-to 10-fold more iron than control cells ( Figure S6). In principle, the de novo proteins could rescue Dfes either by functioning as an esterase or by some alternative mechanism. For example, the de novo proteins could enable iron acquisition by being transported into the media, binding iron with high affinity, being re-transported back into the cell, and then releasing the tightly bound iron intracellularly. Although possible, this alternative mechanism seems unlikely for novel proteins that occur so frequently in a semi-random library.

Irrespective of whether the de novo proteins rescue auxotrophs by providing the same function as the deleted enzyme or by some alternative mechanism, we expected the artificial sequenceswhich were neither selected by evolution (in vivo or in vitro), nor explicitly designed for enzymatic activity -to have far lower levels of activity than naturally evolved sequences. To estimate this level of activity in vivo, we compared the growth rates of deletion strains expressing a high level (400X) of Syn-IlvA-1 to the same strain expressing a low level (1X) of the natural protein, encoded by IlvA. These experiments showed that even when the de novo protein is expressed at ,400-fold higher levels than the natural protein, cells grow much more slowly. (Growth rates are shown in Figure S7.) As expected, the de novo protein exhibits a very low level of biological activity.

Activity levels that are barely sufficient to sustain slow cell growth may not be detectable in vitro. Indeed, others have reported that although overexpression of one E. coli protein can sometimes rescue the deletion of another protein in vivo, these 'moonlighting' activities can be so low that they cannot be detected in vitro [21,22]. Despite these concerns, we attempted to assay biochemical activities in vitro using both cell lysates and purified proteins. Lysates are easy to prepare and are more likely to contain molecular partners (proteins, cofactors, or metals) present in vivo. The disadvantage of lysates, however, is that background activities may obscure the low-level activities of the de novo proteins. We assayed for phosphoserine phosphatase, enterobactin esterase, threonine deaminase, and citrate synthase activities in lysates from cells expressing the respective de novo proteins. In several cases, activity was observed, however, lysates from control cells also showed low levels of activity. This was not surprising, particularly for the Fes and SerB activities, since previous work in our laboratory showed that E. coli lysates contain nonspecific esterases and phosphatases [13].

We also purified several of the de novo proteins. (To avoid contamination by the natural enzyme, purifications were from strains deleted for the natural gene.) We tested these purified proteins for the enzymatic activities deleted in the respective auxotrophs, but were unable to detect activity that was reproducibly above the controls. There are several reasons why such experiments might not demonstrate activity: (i) As noted above, the novel proteins have extremely low levels of activity. (ii) The de novo proteins may require cofactors. We do not know which cofactors might be required, as the novel proteins may use different cofactors than the natural enzymes. (iii) The novel proteins may function by partnering with an E. coli protein. For example, the specificity of an endogenous hydrolase might be altered by binding one of our helical bundles. If this were the case, activity would not be observed in preparations containing only the purified de novo protein. (iv) The Syn protein may function by a different mechanism than the deleted protein, and novel activities would not be detected in experiments designed to assay the natural enzyme.

While we do not yet know the precise mechanisms by which the novel proteins rescue these four deletions in E. coli, we have ruled out several alternative mechanisms including (i) bypass pathways, (ii) activation of known endogenous suppressors, and (iii) induction of a generic stress response (see above). The slow growth rates of cells relying on the novel proteins for survival indicate that regardless of the actual mechanism, the de novo proteins have very low levels of activity -as expected for sequences that were neither designed nor evolved for function. Irrespective of whether the de novo proteins catalyze the same biochemical reaction as the deleted natural protein, or function by some alternative mechanism (and whether they act directly or through interactions with endogenous proteins), elucidating the molecular basis of auxotroph rescue will enhance our understanding of the minimal functions necessary for cell growth.

Replacement of 0.1% of the E. coli genome

We showed above that artificial genes enable cell viability. Next, to assess whether portions of a genome can be replaced by genetic information devised in the laboratory, we probed the ability of several novel sequences to rescue the deletion of several genes simultaneously. A quadruple knockout strain -Dfes DgltA DilvA DserB -was constructed and designated QUAD. This strain has severe growth defects: While the parent strain forms colonies after overnight incubation on either nutrient-rich or nutrientpoor media, QUAD requires almost two days to form colonies on rich media, and as expected, does not form colonies on minimal media. To assess the ability of artificial genes to substitute simultaneously for the deleted functions, we transformed QUAD with derivatives of the pQLinkN vector [23] co-expressing four artificial genes shown above to rescue the four single-gene knockouts. The sequences used for this experiment were syn-fes-2, syn-gltA-1, syn-ilvA-1, and syn-serB-2 ( Figure 3). Transformed cells were plated on minimal media. As a control, we transformed QUAD with empty vector; no colonies were observed for the control. However, as shown in Figure 5, transformation with the vector expressing all four artificial genes produced colonies. As discussed above, the individual Syn proteins are considerably less active than the deleted natural proteins. Therefore, it is not surprising that the simultaneous rescue of four deletions by four artificial proteins produced small colonies that required 13 days to appear. Nonetheless, the rescue of QUAD demonstrates that novel sequences bearing no similarity to naturally evolved proteins can compensate for deletion of a portion of the E. coli chromosome.

The E. coli genome contains approximately 4,000 genes [5]. Of these, only 290 are essential for survival under all conditions [24]. An additional 107 are required for growth in the nutrient-poor media provided by M9 salts with glucose [17]. Thus, only ,400 genes are essential for growth in minimal media. Our results demonstrate that at least 1% of the E. coli genome that is absolutely required for growth under minimal conditions -and 0.1% of the entire E. coli genome -can be replaced by artificial genes designed de novo.

Conclusion

We have demonstrated that sequences designed de novo can provide functions necessary to sustain the growth of living cells. It should be emphasized that these macromolecules were isolated with relatively high frequency from a collection of sequences that were designed to adopt a stable globular fold [7,8,19,20,25], but were not explicitly designed for function.

Both in terms of linear sequence and 3-dimensional structure, the novel proteins differ substantially from the natural proteins they replace: The binary patterned sequences in Figure 3 show no significant sequence similarity to any known proteins. At the structural level, the 102-residue 4-helix bundles specified by our design are much smaller and simpler than the structures of the four natural proteins deleted in the auxotrophs. (Structures are shown in Figure S8). Thus, although natural proteomes include diverse structures and topologies, our studies show that in some cases, cell growth can be sustained by much simpler structures.

The field of synthetic biology -and the related field of astrobiology -aim to go beyond questions about ''what already exists'' and attempt to probe ''what might be possible?'' The results described here suggest that the toolkit for synthetic biology need not be limited to genes and proteins that already exist in nature. We tested 27 deletion strains using a library of 1.5 million sequences designed to fold into 4-helix bundles. From these, we found 18 artificial sequences (Figure 3 and Figure S2) that compensate for the deletion of 4 different genes, and provide biological functions enabling cell growth. Extension of this work to other deletion strains, using larger libraries, and designs that encode a greater diversity of protein structures can be expected to increase substantially the number of life-supporting functions that can be provided by genes designed de novo. The results described here showing that sequences designed in the laboratory can replace portions of the E. coli genome can therefore be viewed as an initial step toward the construction of artificial genomes.

Materials and Methods

Construction of a library of de novo sequences

The design, construction, and characterization of a library of artificial genes encoding binary-patterned de novo proteins are reported elsewhere [11,12]. The library of gene inserts was digested with NdeI and BsrGI, as was plasmid pCA24NMAF2. Double-digested plasmid backbone was gel purified, and the library insert and vector backbone were ligated. Ligation products were transformed into electrocompetent XL1-blue (Stratagene) or MDS42recA (Scarab Genomics, LLC). Colonies were harvested by scraping from LB plates supplemented with 34 mg ml -1 chloramphenicol (cam), and plasmid DNA was purified from the resulting cell suspension. Aliquots from this library of plasmids were used to transform the auxotrophs. The size of this libraryapproximately 1.5610 6 different sequences-was estimated by plating serial dilutions of the transformations of the initial ligation products on LB-cam [12].

Isolation of de novo sequences that rescue auxotrophs

The collection of plasmids encoding our library of novel sequences, and two negative control plasmids, pCA24N-lacZ and pCA24NMAF2 (empty vector), were used to transform 27 E. coli auxotrophs. These were chosen from Keio strains that grow on LB, but not on M9-glucose. Following electroporation, cells were outgrown at 37uC, harvested by centrifugation, washed and resuspended in chilled M9 salts, and spread on both LB supplemented with 30 mg ml 21 kanamycin (kan), 34 mg ml 21 cam; and on M9-glucose supplemented with 50 mM IPTG, 30 mg ml 21 kan, 34 mg ml 21 cam. After several days at 33uC, plates were scored for number of colonies. In addition, a 1:100 dilution of library-transformed cells was spread onto LB-cam plates to measure transformation frequency under conditions that were non-selective for auxotroph growth.

Growth assays

Single colonies were inoculated into LB (30 mg ml 21 kan, 170 mg ml 21 cam). Following overnight growth at 37uC, 1 ml of each culture was harvested by centrifugation at 4uC. Cell pellets were washed and resuspended in 1 ml chilled M9 salts, and the resulting cell suspensions were diluted 100-fold into M9-glucose (50 mM IPTG, 30 mg ml 21 kan, 34 mg ml 21 cam). Cultures were grown at 33uC and assayed by measuring absorbance at 600 nm.

Additional methods are described in Supporting Information Text S1.

Supporting Information

Text S1 Supplementary materials and methods. (DOC) Both exhibit a 4 amino acid insertion into helix 1, and a 1 amino acid deletion from helix 4. Syn-IlvA-5 is a 106-residue protein, with an 11 amino acid insertion into the second helix and a 7 amino acid deletion from helix 4. These spurious mutations occur at the DNA level and are due either to errors in oligonucleotide synthesis or rare instances of the mis-assembly of library insert building blocks. As long as such spurious mutations do not affect frame or introduce stop codons, the mutated segments survive preselection, and as shown by our results, can contribute to the synthesis of productive sequences. (B) Growth curves for the DilvA strain expressing designed artificial proteins with spurious mutations. The bottom curve shows that the negative control expressing LacZ (open circles) does not enable growth. In contrast, the positive control expressing IlvA (closed circles) grows well. Like the novel sequences that adhere precisely to the design, the designed proteins with spurious mutations enable growth that is well above background. Growth curves correspond to the sequences in A as follows: Syn-IlvA-3, closed squares; Syn   Figure S4 Circular dichroism spectra. CD spectra demonstrate that purified de novo proteins fold into alpha-helical structures. Cultures of E. coli were grown to an OD600 of approximately 0.6 and induced with IPTG. Protein was extracted from the cells by repeated cycles of freezing and thawing. Purification of the Syn proteins to 95% (coommaise-stained SDS-PAGE) was achieved by cation exchange chromatography. Proteins were dialyzed into phosphate buffer for CD analysis.  Figure S6 Assay for iron using inductively coupled plasma (ICP) spectrometry. The Dfes strain was transformed with a plasmid directing the high level expression of either LacZ or one of the artificial sequences that rescued the fes deletion. Cells were grown in LB, harvested, and tested for iron accumulation. The artificial proteins enable the accumulation of 6-10 fold more iron than the control. (TIFF) Figure S7 De novo proteins are considerably less active than the natural proteins they replace. (A) Growth on minimal media of DilvA cells expressing different sequences. Cells expressing the natural IlvA protein grow well either in the presence (Blue) or absence (Red) of IPTG. In contrast, cells expressing Syn-IlvA-1 grow significantly more slowly, even in the presence of IPTG (Green). [These cells do not grow in the absence of IPTG.] Control cells expressing LacZ fail to grow even in the presence of IPTG (Purple). All proteins were expressed off the pCA24N plasmid. (All curves except the red curve represent experiments done in the presence of 0.5 mM IPTG.) (B) Beta-galactosidase assays demonstrate that IPTG induces 402 (+/27) fold increase in protein expression. LacZ was expressed from the pCA24N plasmid, either in the presence or the absence of IPTG, and levels of beta-galactosidase (Y-axis) were assayed according to standard procedures. The data summarized in this figure show that the Syn-IlvA protein is substantially less active than natural IlvA. The beta-galactosidase assays in panel B demonstrate that IPTG causes a 402 (+/27) fold increase in protein expression. The results shown in panel A show that for the natural IlvA protein, this change in expression level has no effect on the rate of cell growth: Even low level expression in the absence of IPTG is sufficient for the natural protein to sustain robust growth. In contrast, cells that depend on Syn-IlvA grow slowly even when expression is induced 400-fold. Since cells relying on 400x levels of Syn-IlvA require significantly more time to grow than cells expressing 1x levels of the natural protein, the artificial protein is ,1/400 as active as the natural protein. These experiments demonstrate that the artificial proteins are substantially less active in their ability to sustain cell growth. However, they do not indicate that the artificial proteins function by the same mechanism as the natural protein. Indeed, the reduced ability of the artificial proteins to sustain cell growth may be due to their functioning by a less effective mechanism. (TIFF)

Figure 1 .
1Design of a collection of novel proteins and rescue of E. coli auxotrophs.

Figure 2 .
2Rescue of E. coli auxotrophs by de novo proteins. (A) E. coli strain BW25113 Dfes was transformed with a control plasmid overexpressing LacZ (left half), or with a library of plasmids encoding the collection of novel proteins (right half). Cells were plated on M9-glucose supplemented with 50 mM IPTG, 30 mg ml 21 kanamycin, and 34 mg ml -1 chloramphenicol. Cells transformed with the control plasmid fail to grow, whereas cells transformed with the library yield occasional colonies. (B) Similar results for DilvA. (Similar results were also observed for DserB and DgltA -data not shown.) (C) Re-transformation of DilvA cells with a single sequence, syn-ilvA-1. A similar number of colonies grow on M9-glucose (left) as on LB (right). Results for other sequences were similar to those shown in (C), and are summarized in Figure S1. doi:10.1371/journal.pone.0015364.g002

Figure 3 .
3Designed amino acid sequences that enable growth of E. coli auxotrophs on selective media. Names of the novel (Syn) proteins and the auxotroph rescued are listed at left. The novel sequences follow the designed binary pattern of polar (red) and nonpolar (yellow) residues. The top line shows the template designed to encode four amphiphilic alpha-helices. Combinatorial positions are shown as empty circles (polar residues), filled circles (nonpolar residues), or hyphens (turn residues). Sequences were submitted to the European Nucleotide Archive and assigned accession numbers FR718891 -FR718908. doi:10.1371/journal.pone.0015364.g003

Figure 4 .
4Growth of auxotrophic strains of E. coli in selective liquid media. In each panel, the bottom curve shows that the negative control expressing LacZ (open circles) does not support growth. In contrast, most of the positive controls (black circles) expressing the natural E. coli proteins grow well. (Fes is an exception because overexpression of E. coli Fes is toxic.) The designed proteins enable growth that is well above background. (A) DserB: LacZ, open circles; SerB, closed circles; Syn-SerB-1, closed triangles; Syn-SerB-2, open triangles; Syn-SerB-3, closed squares; and Syn-SerB-4, open squares. (B) DgltA: LacZ, open circles; GltA, closed circles; and Syn-GltA-1, closed triangles. (C) DilvA: LacZ, open circles; IlvA, closed circles; Syn-IlvA-1, closed triangles; and Syn-IlvA-2, open triangles. (D) Dfes: LacZ, open circles; Fes, closed circles; Syn-Fes-1, closed triangles; Syn-Fes-2, open triangles; Syn-Fes-3, closed squares; Syn-Fes-4, open squares; Syn-Fes-5, closed diamonds; Syn-Fes-6, open diamonds; Syn-Fes-7, 6; and Syn-Fes-8, +. doi:10.1371/journal.pone.0015364.g004

Figure S1
S1Reconfirmation of hits. Colony counts for retransformation of DilvA cells with several different hits. The histogram summarizes 13 experiments of the type shown in figure 2C. The number of colonies on M9-glucose minimal media (dark blue) is similar to the number on LB rich media (light blue). (TIFF) Figure S2 De novo sequences with spurious mutations and corresponding DilvA growth curves. (A) Novel sequences with spurious mutations. Red indicates polar residues, yellow indicates nonpolar residues, and turns are in blue. Syn-IlvA-3 and Syn-IlvA-4 are 105-residue proteins differing by a single residue at position 28.

Figure S3
S3Reactions and pathways of the deleted proteins. (A) serB encodes phosphoserine phosphatase, responsible for the final step in serine biosynthesis. (B) ilvA encodes biosynthetic threonine deaminase, which catalyzes the first step in the production of isoleucine from threonine. (C) fes encodes enterobactin esterase, which cleaves iron-bound enterobactin. (D) gltA encodes citrate synthase, which catalyzes an early step in glutamate biosynthesis. (TIFF)

Figure 5 .
5Rescue of a quadruple knockout E. coli by co-expression of 4 de novo proteins. (A) Schematic of knockout rescue. Top: Wild-type E. coli transformed with empty vector forms colonies on M9/glucose minimal media. Middle: QUAD E. coli transformed with empty vector does not form colonies on minimal media. Bottom: QUAD transformed with a vector co-expressing four de novo sequences forms colonies on minimal media. (B) Colonies are not observed when QUAD is transformed with empty vector (top panel), but are observed when transformed with a vector expressing four artificial genes (bottom panel). Scale bar = 1 mm. doi:10.1371/journal.pone.0015364.g005

Figure S5
S5Mutation of Lys42 to Ala destroys the ability of a synthetic protein to rescue DilvA. (A) Sequence alignment of the six synthetic proteins that rescue DilvA. Two charged residues are conserved, E and K (red arrows). Green lines mark regions that are conserved in the designed template. (B) Transformation of DilvA cells with Syn-IlvA-1 and Syn-IlvA-1-K42A. Cells expressing Syn-IlvA-1 grow on M9-glucose, but cells expressing Syn-IlvA-1-K42A do not. (C) SDS-PAGE of protein levels after induction with IPTG. Levels of Syn-IlvA-1-K42A are approximately equivalent to those of Syn-IlvA-1. Red arrow marks the band corresponding to the synthetic protein. (TIFF)

Figure S8
S8Structures of the 4 natural proteins compared with protein S-824, a de novo 4-helix bundle from a binary patterned library. Coordinates are from the PDB. (A) M. jannaschii SerB (1f5s). (B) E. coli GltA (1k3p). (C) The asymmetric unit of E. coli IlvA (1tdj). (D) S. flexneri Fes (3c87). (E) De novo protein S-824 (1p68). (TIFF)

Table 1 .
1Complementation frequency and time required for colony formation on selective media (M9-glucose). DargB, DargE, DargH, DaroB, DaroC, DaroD, DaroE, DbioA, DcysG, DglnA, DhisA, DilvC, DilvD, DleuB, DleuC, DleuD, DpdxA, Dppc, DproB, DpurF, DpyrF, DthrB, and DthrC. doi:10.1371/journal.pone.0015364.t001Complementation
Frequency

Time to
Grow (Days)

DserB
,1/50,000
4

DgltA
,1/1,000,000
4

DilvA
,1/3,000
3

Dfes
,1/10,000
2

Novel proteins capable of enabling cell growth were isolated for 4 strains.
Complementation frequency was calculated by dividing the number of colonies
on selective plates (rescued transformants) by the number of colonies on rich
plates (total transformants). This apparent frequency was then adjusted to
estimate the true complementation rate by correcting for any false positives
that failed to restreak or retransform. The following additional strains from the
Keio collection were tested, but were not rescued by sequences from the
library:
PLoS ONE | www.plosone.org
January 2011 | Volume 6 | Issue 1 | e15364
AcknowledgmentsWe thank Wayne Patrick and Ichiro Matsumura for providing materials and advice at the outset of the project; Natividad Ruiz and Tom Silhavy for providing access to the Keio collection, and for guidance about creating the multiple knockouts; Joshua Lees and Fred Hughson for advice on the pQLinkN vector; Josh Rabinowitz and John Groves for advice; and Izhack Cherny for advice and encouragement.Author Contributions
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Hybrid selection for sequencing pathogen genomes from clinical samples

Alexandre Melnikov
Kevin Galinsky
Peter Rogov
Timothy Fennell
Daria Van Tyne
Carsten Russ
Rachel Daniels
Kayla G Barnes
James Bochicchio
Daouda Ndiaye
Papa D Sene
Dyann F Wirth
Chad Nusbaum
Sarah K Volkman
Bruce W Birren
Andreas Gnirke
Daniel E Neafsey
Hybrid selection for sequencing pathogen genomes from clinical samples
M E T H O D Open Access
We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla.

Background

The falling cost of DNA sequencing means that sample quality, rather than expense, is now the blocking issue for many infectious disease genome sequencing projects. Pathogen genomes are generally very small relative to that of their human host, and are typically haploid in nature. Therefore, even a modest number of nucleated human cells present in infectious disease samples may result in the pathogen DNA representation being dwarfed relative to the host human DNA. This difference in representation poses a significant challenge to achieving adequate sequence coverage of the pathogen genome in a cost-effective manner. Separation of host and pathogen cells prior to DNA extraction can be difficult or inconvenient, particularly in field settings common to clinics in developing countries.

This barrier to the efficient sequencing of pathogen genomes comes at a time when the potential motivations and rewards for large-scale sequencing of pathogens are becoming increasingly clear. Examples abound to demonstrate how whole-genome analyses of pathogen population structure from large numbers of isolates can help to identify the source of disease outbreaks or hidden subpopulations. Whole genome sequencing of 35 Salmonella enterica samples was recently performed by the United States Food and Drug Administration in order to identify the source of a foodborne illness outbreak that affected approximately 300 individuals in 2009 and 2010 [1]. Whole genome sequencing of 20 isolates of pathogenic Coccidiodies spp. fungi identified gene flow in select genomic regions between the recently diverged Coccidiodies immitis and Coccidiodies posadasii [2]. Whole genome sequencing and comparative SNP analysis of unculturable Mycobacterium leprae isolates was utilized to demonstrate that a third of leprosy infections in the United States derive from armadillos [3]. So-called 'third generation' sequencing was successfully employed to identify the origin of the recent Haitian cholera outbreak strain via de novo sequencing of 5 isolates and comparison of those sequences to 23 previously sequenced isolates of Vibrio cholera [4]. In addition, the increasing use of genomewide association studies to determine the genetic basis of important infectious disease phenotypes, such as drug resistance in malaria parasites [5,6], will require sequencing or genotyping hundreds to thousands of pathogen isolates, making a shortage of quality specimens an acute problem. All of these studies could have been performed more expediently if a culturing step were not required to eliminate DNA derived from the host or environment.

Existing methods for dealing with DNA contamination in infectious disease samples typically require significant time, money, and/or special handling of samples at the time of collection. Taking Plasmodium falciparum as a model case, malaria parasite samples in blood may be adapted to in vitro culture and sustained in a pure medium of DNA-free human red blood cells. The adaptation process, however, can take more than 6 weeks, requires considerable expertise and expense [7], and may potentially select for culturable variants. To remedy this, DNA-containing white blood cells may be depleted directly from malaria patient blood samples prior to cell lysis via differential density centrifugation or column filtration [8][9][10]. While depletion methodologies may reduce white cell abundance to levels useful for biochemical assays, the 100-fold disparity in genome size between human and malaria means that an even modest number of host cells can compromise a sample for genome sequencing. In addition, white blood cell depletion currently requires a significant volume of blood to be drawn from patients (approximately 5 ml), and the blood must then be stored at minus 70°C in a special medium to preserve cellular integrity. This could preclude sample collection for genome sequencing from many clinical trials due to protocol limitations or lack of equipment in the field. Furthermore, pathogens that infect or closely associate with nucleated host cells, such as Plasmodium vivax, Trypanosoma cruzi, or Chlamydia trachomatis, are not amenable to purification by white cell depletion. Endosymbionts such as Wolbachia, which influence host fertility and other traits in filarial worms, insect disease vectors, and diverse other taxa, may only be cultured in an intracellular system [11], precluding easy isolation of their genomic DNA for sequencing except by elaborate methods [12].

To address this problem we have adapted a solution hybrid selection approach originally developed for the purification of resequencing targets in the human genome [13]. In brief, biotinylated RNA probes complementary to the pathogen genome ('baits') are hybridized to pathogen DNA in solution and pulled down with magnetic streptavidin-coated beads. Host DNA is washed away and the captured pathogen DNA may then be eluted and amplified for sequencing or genotyping. We experimented with two approaches to bait design: synthetic 140-bp oligos targeting specific regions of the P. falciparum 3D7 reference genome assembly and 'whole genome baits' (WGBs) generated from pure P. falciparum DNA. Using this protocol, we achieved significant enrichment of P. falciparum DNA, to a level that allowed us to conduct whole genome sequencing on samples that otherwise would have been prohibitively expensive to sequence.

Results and discussion

Hybrid selection on a mock clinical malaria sample

We performed hybrid selection with both classes of bait on a mock clinical sample consisting of 99% human DNA and 1% Plasmodium DNA by mass, which falls within the range of DNA ratios found in many malaria clinical samples (Table 1). Hybridization and washing steps (see Materials and methods) were carried out under standard high stringency conditions to reduce capture of host DNA. The hybrid selection protocol requires a minimum of 2 μg of input DNA (combined host and pathogen), a quantity that may not be available from many types of field samples. Therefore, we also performed hybrid selection with both bait classes on 2 μg of whole genome amplified DNA generated from 10 ng of the mock clinical sample. Quantitative PCR (qPCR) analysis indicated that whole genome amplification (WGA) does not significantly alter the fraction of malaria DNA present in the sample (post-WGA percentage P. falciparum DNA = 1.1 ± 0.1).

Sequencing of the hybrid-selected samples revealed a significant increase in representation of Plasmodium DNA in every case. The synthetic baits respectively yielded an average of 41-fold and 44-fold parasite DNA enrichment for unamplified and WGA simulated clinical samples in genomic regions targeted by the baits, as measured by qPCR. Whole genome baits yielded parasite genome-wide average enrichment levels of 37-fold and 40-fold for the unamplified and WGA input samples, respectively.

Illumina sequencing coverage in the WGB hybridselected samples is correlated with GC content, mirroring what is observed in sequencing data from pure P. falciparum DNA (Figure 1a). With a genome-wide A/T composition of 81% [14], achieving uniform sequencing coverage of the P. falciparum genome is challenging even under ideal circumstances. Despite this challenge, we observed no reduction in coverage uniformity as a result of the hybrid selection process. WGA did not compromise mean genome-wide sequencing coverage relative to unamplified input DNA (67.5 × versus 67.1 × for a single Illumina GAIIx lane, respectively). Sequencing coverage of the samples hybrid selected using synthetic 140-bp baits was tightly localized to the genomic regions to which baits were designed ( Figure 1b). Coverage levels in baited regions were significantly higher than the levels observed from comparable sequencing of pure P. falciparum DNA (mean coverage = 143.8 × and 92 ×, respectively; Wilcoxon rank sum test, W = 6.7E12, P < 2.2e-16). This indicates that hybrid selection with synthetic baits may be useful not only for reducing offtarget coverage in the host genome, but also for strategically augmenting coverage levels in regions of pathogen genomes where heightened sequence coverage could be informative, such as highly polymorphic antigenic regions subject to host immune pressure.

Though effective sequencing coverage levels are reduced in the hybrid-selected mock clinical samples relative to pure P. falciparum DNA due to the incomplete elimination of human DNA, this reduction is small compared to the 100-fold reduction in coverage expected without hybrid selection. Genome-wide coverage is depicted in Figure 2a, which illustrates that the extent of the genome covered to various thresholds is highly similar for the pure P. falciparum and hybridselected mock clinical samples, and significantly higher than simulated coverage levels we would have predicted to have observed from sequencing an un-purified version of the sample. Genome-wide coverage levels as a function of the local %GC (the percentage of nucleotides in the genome that are G or C; %G+C) are plotted in Figure 2b for the WGB experiments. The relationship between %GC and coverage observed in whole genome shotgun sequencing data is decreased by hybrid selection due to reduced coverage in rare high %GC genomic regions (Spearman's r s for %GC versus coverage of pure malaria DNA, 0.86; versus WGB hybrid-selected DNA, 0.59; versus WGA + WGB hybrid-selected DNA, 0.64). The vertical line in Figure 2b represents the average % GC of exonic sequence (23%). Assuming a minimum threshold of 10-fold sequencing coverage is required for accurate SNP calling, 99.2% of exonic bases exhibited this coverage or greater in reads generated from the pure P. falciparum DNA sample. The unamplified and amplified hybrid-selected samples achieved at least 10fold coverage for 98.3% and 98.0% of exonic bases, respectively. Given that previous pathogen population genomic analyses of outbreaks or population structure have been SNP-based [1,2,4], this indicates that sequencing data generated from hybrid-selected clinical samples could be as useful as data generated from pure pathogen DNA samples for downstream analyses.

Further comparison of sequencing coverage between hybrid-selected and pure P. falciparum DNA indicates that local %GC and polymorphism rate do not significantly influence sequencing coverage in a hybridselected sample (Additional file 1).

We attempted to optimize our hybrid selection protocol by exploring two different hybridization temperatures (60°C versus 65°C) and four different 10-minute wash stringencies (0.1 × SSC, 0.25 × SSC,0.5 × SSC, and 0.75 × SSC). Eight mock clinical samples were hybridized with WGB and washed under all combinations of the above conditions. Enrichment was measured by qPCR and sequencing (one indexed Illumina GAIIx lane). We observed the best enrichment under the standard high stringency conditions used for all previously reported experiments (hybridization at 65°C and high stringency wash (0.1 × SSC). Results are presented in Table 2.

In summary, both bait strategies performed effectively and now offer investigators a method to sequence either targeted regions or complete genomes of pathogens in clinical samples dominated by host DNA. Pairing this hybrid selection protocol with WGA further expands the range of clinical samples now eligible for efficient pathogen genome sequencing. For example, for Plasmodium it should now be possible to sequence the parasite genome directly from dried blood spots on filter paper, an easily collectable and storable sample format.

Hybrid selection on authentic clinical samples

To test this application, we performed WGA and hybrid selection on DNA extracted from a clinical P. falciparum sample (Th231.08) collected on filter paper in Thies, Senegal in 2008 and stored at room temperature for over a year. By qPCR we estimated the Plasmodium   DNA in the original sample to comprise approximately 0.11% of the total DNA by mass. Following WGA and hybrid selection, Plasmodium DNA represented 7.7% of total DNA present, an approximately 70-fold increase in parasite DNA representation. Illumina HiSeq sequencing data confirmed that at least 5.9% of mappable reads in the hybrid-selected sample corresponded to Plasmodium. The fraction of human reads after hybrid selection remained high due to the extreme initial ratio of host: parasite DNA, but the enrichment factor in this case was sufficient to rescue the feasibility of sequencing this sample. We evaluated the accuracy and utility of the data by calling SNPs against the P. falciparum reference assembly. We identified a total of 26,366 SNPs relative to the P. falciparum reference assembly (more than one per kilobase), close to the number of SNPs identified (33,094 to 41,123) from 11 other culture-adapted Senegalese parasite lines sequenced without hybrid selection. Further SNPs could likely be discovered by further augmenting coverage. While the depth of coverage we obtained from this experiment would not be sufficient for de novo genome assembly, SNP calling against a reference assembly is the end-stage analysis for most Illumina data (for example, [1][2][3][4]) and therefore a good indication of a dataset's potential utility. Principal components analysis of SNP genotypes confirms the similar genomic profile of the hybrid-selected and non-hybrid-selected Senegalese strains, as well as hybrid-selected and non-hybridselected 3D7 reference strain datasets generated from sequencing the mock clinical samples (Figure 3). Despite the use of WGBs generated from the 3D7 reference genome, the DNA captured from the Senegal isolate has the SNP profile of Senegal DNA, rather than 3D7 DNA, suggesting that polymorphisms do not strongly bias enrichment. In addition, the highly polymorphic regions of the isolate did not suffer a relative drop in sequencing coverage after hybrid selection. Selection of a panel of 12 other clinical malaria samples from Senegal yielded an average of 35-fold enrichment, as measured by qPCR (Table 1), with enrichment amount inversely proportional to the initial fraction of parasite DNA in the samples.

We conducted a second round of hybrid selection on the Th231.08 clinical sample to determine whether the Plasmodium DNA titer in the sample could be boosted above approximately 7%. The second round of hybrid selection was carried out under identical hybridization and wash conditions. qPCR analysis indicates this yielded a sample in which 47.5% of the genetic material was Plasmodium by mass (a 6.7-fold enrichment). This lower fold enrichment is consistent with our previous observation that fold enrichment is inversely proportional to initial parasite DNA titer, but in this case an additional round of hybrid selection yields a sample even more amenable to cost-efficient and deep sequencing.

Although sequencing has become considerably less expensive in recent years, it remains financially impractical  Figure 3 Principal component analysis plot based on SNP calls produced from hybrid-selected and non-hybrid-selected samples. The hybrid-selected clinical sample from Senegal (red) clusters with 12 previously sequenced Senegal samples (blue). The hybrid-selected 3D7 samples (red) cluster with the non-hybridselected 3D7 sample (yellow). P. falciparum isolates from India (purple) and Thailand (brown) are also represented. PC, principal content.

to sequence pathogen genomes from clinical samples at scale due to the gross excess of host DNA typically present. The simplest way to compensate for host DNA contamination is to augment sequencing coverage depth. However, this strategy can be costly for all but the most lightly contaminated samples. In contrast, the cost of purification by hybrid selection using whole genome baits is approximately US$250, which is roughly equivalent to the current cost of generating 20-fold coverage of the 23 Mb P. falciparum genome from pure template using a fraction of an Illumina HiSeq lane. For augmented coverage to be an affordable strategy relative to hybrid selection for a target coverage level of 40 × in a genome of this size, samples must contain at least 50% pathogen DNA. This titer of parasite DNA is rarely found in clinical samples unless white cell depletion is performed prior to DNA extraction. For a more typical clinical sample containing only 1% P. falciparum DNA, hybrid selection resulting in 40-fold enrichment enables 40 × coverage depth for a dramatically lower total price (approximately $1,000) than deeper sequencing of the unpurified sample (approximately $40,000).

Conclusions

The modest cost and high performance of this hybrid selection purification protocol will facilitate sequencing of archival clinical samples of malaria parasites and other pathogens previously considered unfit for sequencing by any methodology. This may enable sequencing of important samples stored on filter papers or diagnostic slides predating the spread of drug resistance or associated with historic outbreaks. This purification protocol also broadens the accessibility of sequencing for clinical samples of infectious organisms for which in vitro culture is possible but costly or inconvenient, such as class IV 'select agents' recognized by the Centre for Disease Control. This protocol is not limited to pathogens, and should be equally useful in sequencing commensal or symbiotic organisms closely associated with their host, such as intracellular Wolbachia bacteria, as was recently demonstrated by Kent et al. in their application of an array-based capture protocol [15]. The reduction in sample quality and quantity requirements permitted by hybrid selection will simplify protocol design in future large-scale clinical studies and help realize the benefits of inexpensive, massively parallel sequencing technologies for studying infectious diseases in diverse contexts.

Materials and methods

Samples

Mock clinical samples were generated by mixing Homo sapiens NA15510 DNA with a pure preparation of P. falciparum 3D7 parasite DNA at a ratio of 99:1 (H. sapiens: P. falciparum) by mass. Samples were fluorescently quantified prior to mixing using a PicoGreen [16] assay. Authentic clinical samples were collected in 2008 from symptomatic patients at a clinic in Thies, Senegal under an approved institutional review board protocol. Samples consisted of whole blood dried and stored on a Whatman FTA card (fast technology for analysis of nucleic acids) and/or frozen whole blood stored in glycerolyte 57 solution. DNA was extracted using a DNeasy kit (Qiagen Hilden, North Rhine-Westphalia, Germany).

Whole frozen blood samples yielded sufficient DNA for hybrid selection, but samples from FTA cards typically yielded less than 100 ng of DNA and required WGA. WGA was performed using the Repli-G kit (Qiagen).

Bait design and preparation

Synthetic 140-bp oligos were obtained from Agilent and designed to capture exonic regions of the P. falciparum genome as defined in the 3D7 v.5.0 reference assembly. The final bait set included 24,246 oligos (3.4 Mb) with unique BLAT matches to the P. falciparum 3D7 reference genome assembly and no homology to the human genome. Baits and locations are listed in Additional file 2. To generate synthetic single-stranded biotinylated RNA bait, in vitro transcription was performed with biotin-labeled UTP using the MEGAshortscript T7 kit (Ambion Austin, Texas, United States) as described previously [13]. WGB was generated at the Broad Institute. For input, 3 μg of P. falciparum 3D7 DNA was sheared for 4 minutes on a Covaris E210 instrument set to duty cycle 5, intensity 5 and 200 cycles per burst. The mode of the resulting fragment size distribution was 250 bp. End repair, addition of a 3'-A, adaptor ligation and reaction clean-up followed the Illumina's genomic DNA sample preparation kit protocol except that adapter consisted of oligonucleotides 5'-TGTAACATCACAGCATCACCGC CATCAGTCxT-3' ('x' refers to an exonuclease I-resistant phosphorothioate linkage) and 5'-[PHOS]GACTG ATGGCGCACTACGACACTACAATGT-3'. The ligation products were cleaned up (Qiagen), amplified by 8 to 12 cycles of PCR on an ABI GeneAmp 9700 thermocycler in Phusion High-Fidelity PCR master mix with HF buffer (NEB Ipswich, Massachusetts, United States) using PCR forward primer 5'-CGCTCAGCGGCCG CAGCATCACCGCCATCAGT-3' and reverse primer 5'-CGCTCAGCGGCCGCGTCGTAGTGCGCCATCAGT-3' (ABI Carlsbad, California, United States). Initial denaturation was 30 s at 98°C. Each cycle was 10 s at 98°C, 30 s at 50°C and 30 s at 68°C. PCR products were sizeselected on a 4% NuSieve 3:1 agarose gel followed by QIAquick gel extraction. To add a T7 promoter, sizeselected PCR products were re-amplified as above using the forward primer 5'-GGATTCTAATACGACTCAC TATACGCTCAGCGGCCGCAGCATCACCGCCAT CAGT-3'. Qiagen-purified PCR product was used as template for whole genome biotinylated RNA bait preparation with the MEGAshortscript T7 kit (Ambion) [13].

Hybrid selection

Hybrid selection using either synthetic bait or WGB was carried out as described previously [13]. Hybridization was conducted at 65°C for 66 h with 2 μg of 'pond' libraries carrying standard or indexed Illumina paired-end adapter sequences and 500 ng of bait in a volume of 30 μl. After hybridization, captured DNA was pulled down using streptavidin Dynabeads (Invitrogen Carlsbad, California, United States). Beads were washed once at room temperature for 15 minutes with 0.5 ml 1 × SSC/0.1% SDS, followed by three 10-minute washes at 65°C with 0.5 ml pre-warmed 0.1 × SSC/ 0.1% SDS, re-suspending the beads once at each washing step. Hybrid-selected DNA was eluted with 50 μl 0.1 M NaOH. After 10 minutes at room temperature, the beads were pulled down, the supernatant transferred to a tube containing 70 μl of 1 M Tris-HCl, pH 7.5, and the neutralized DNA desalted and concentrated on a QIAquick MinElute column and eluted in 20 μl.

Quantitative PCR enrichment measurement

Enrichment of malaria DNA in samples was assessed using a panel of malaria qPCR primers designed to conserved regions of the P. falciparum 3D7 v.5.0 reference genome. Enrichment for each amplicon was calculated as the ratio between the amount of DNA presented preand post-hybrid selection, with threshold cycle (cT) counts corrected for qPCR efficiency using a standard curve for each amplicon. All qPCR reactions utilized 1 μl of template containing 1 ng of total DNA. Estimated enrichment for the samples was calculated as the mean enrichment observed across all tested amplicons. Primer sequences and locations are listed in Additional file 3. Quantification of human DNA in the clinical samples was performed prior to sequencing using the Taqman RNase P Detection Reagents kit (Applied Biosystems Carlsbad, California, United States). bioinformatic analyses, edited the manuscript and reviewed the data. TF performed bioinformatic analyses. DEN performed bioinformatic analyses, supervised the project, conceived and initiated the project and wrote the manuscript. DN and PDS provided samples. DVT, KGB and RD performed DNA extractions on the samples. JB coordinated sequencing. CR and DFW supervised the project. SKV, CN and BWB supervised the project, edited the manuscript and reviewed the data. All authors have read and approved the manuscript for publication.

Figure 1
1Sequencing coverage plots from a randomly chosen region of P. falciparum chromosome 1. (a) Unamplified (green) and WGA (purple) WGBs compared to pure P. falciparum (gray). (b) Unamplified (green) and WGA (purple) synthetic bait read coverage compared to pure P. falciparum (gray). Red bars indicate bait locations. (c) Local %GC (the percentage of nucleotides in the genome that are G or C; in 140-bp windows). Green bars indicate exons. Chr, chromosome.

Figure 2
2Genome-wide sequencing coverage and composition. (a) Coverage thresholds for unamplified (green) and WGA (purple) whole genome baits compared to pure P. falciparum (gray) and simulated coverage from a non-hybrid-selected mock clinical sample (yellow). (b) Genome-wide coverage as a function of %GC. The vertical black line represents average exonic %GC. The red histogram represents the probability density distribution of genome composition (right vertical axis). Lines depict coverage (left vertical axis) of pure P. falciparum DNA (gray), as well as unamplified (green) and WGA (purple) hybrid-selected samples initially containing 1% P. falciparum DNA. %GC, the percentage of nucleotides in the genome which are G or C.

Sequencing
Each sample was sequenced at the Broad Institute using one lane of Illumina 76-bp paired-end reads. The libraries of pure P. falciparum DNA and hybrid-selected artificial clinical samples were each sequenced with one Illumina GAIIx lane. The hybrid-selected authentic clinical sample (Th231.08) was sequenced with one Illumina HiSeq lane. Sequence data have been deposited in the NCBI Short Read Archive under accession number [SRA029706].

Table 1
1Quantitative PCR enrichment measurements from 12 clinical samplesPercentage
Parasite [DNA] (pg/μl)

Sample
parasite DNA
WGA
Pre-hybrid selection a
Post-hybrid selection a
Fold enrichment

Th231.08 (round 1)
0.11
Yes
1.8 (0.6)
71.1 (5.6)
39.7

Th231.08 (round 2)
7.7
No
71.1 (5.6)
349.1 (74.9)
4.9

Th145.08
20
No
198.4 (17.4)
477.6 (66.7)
2.4

Th032.09
12
No
114.7 (2.9)
372.6 (59.3)
3.2

Th029.09
3
No
33.6 (0.8)
317.3 (54.7)
9.4

Th093.09
2.8
No
28.5 (1.5)
365.6 (53.4)
12.8

Th090.08
2.3
No
37.7 (1.1)
300.4 (46.9)
8.0
Th139.08
2.1
No
23.6 (0.6)
346.2 (50.7)
14.7

Th197.08
1.1
No
14.6 (0.0)
222.7 (36.1)
15.3

Th140.08
0.99
No
9.6 (0.1)
251.5 (37.4)
26.2

Th190.08
0.64
No
5.1 (0.2)
218.7 (34.0)
43.2

Th238.08
0.53
No
6.7 (0.2)
273.4 (38.1)
41.0

Th127.09
1.6
No
26.8 (0.4)
368.5 (57.1)
13.7

Th175.08
48
Yes
275.8 (7.2)
556.9 (79.4)
2.0

a

Numbers in parentheses represent standard deviations. WGA, whole genome amplification.

Melnikov et al. Genome Biology 2011, 12:R73
http://genomebiology.com/2011/12/8/R73

Table 2
2Quantitative PCR enrichment measurementsHybrid temperature
Stringency
Wash
Pre-hybrid selection
[DNA] (pg/μl)

Post-hybrid selection
[DNA] (pg/μl)

Fold enrichment

65°C
High
0.10 × SSC
10.0
342.9
34.3

Med/high
0.25 × SSC
10.0
258.2
25.8
Med/low
0.50 × SSC
10.0
227.9
22.8

Low
0.75 × SSC
10.0
181.4
18.1

60°C
High
0.10 × SSC
10.0
288.6
28.9

Med/high
0.25 × SSC
10.0
232.9
23.3

Med/low
0.50 × SSC
10.0
203.5
20.4

Low
0.75 × SSC
10.0
196.3
19.6

Melnikov et al. Genome Biology 2011, 12:R73 http://genomebiology.com/2011/12/8/R73
AcknowledgementsThis project has been funded in part with Federal funds from the National Institute of Allergy and Infectious Diseases National Institutes of Health, Department of Health and Human Services, under contract number HHSN27220090018C. Funding was also supplied by a Global Health Program grant (number 49764) from the Bill and Melinda Gates Foundation and a grant from the National Human Genome Research Institute (number HG03067-05). We thank the Broad sequencing platform for sequence data generation.Competing interestsThe authors disclose that they are seeking to patent this application of hybrid selection and whole genome bait preparation.AnalysisQuality scores on Illumina reads were rescaled using the MAQ sol2sanger utility[17]. Reads were then aligned to P. falciparum 3D7 (PlasmoDB 5.0) using BWA[18]. Sequenced reads were sorted and the consensus sequence was determined using the SAMtools utilities[19]. %GC was calculated from 140-bp windows across the P. falciparum genome.The human:P. falciparum DNA ratio in each sequence dataset was estimated from sequencing data by randomly sampling 50K pairs of mated reads and measuring the fractions that uniquely mapped to human versus P. falciparum reference genome assemblies.Simulated sequencing read coverage for the mock clinical sample prior to hybrid selection was performed by randomly sampling 1% of the read data generated for the pure P. falciparum sample, under the tested assumption that read coverage scales closely with parasite DNA fraction.Principal components analysis was performed using Eigensoft software[20]on 8,300 non-singleton SNPs with coverage of at least 10-fold in all strains and consensus quality scores of at least 30.Additional material
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Hybrid selection for sequencing pathogen genomes from clinical samples. Melnikov, Genome Biology. 1273Cite this article asCite this article as: Melnikov et al.: Hybrid selection for sequencing pathogen genomes from clinical samples. Genome Biology 2011 12:R73.