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# Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome

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Science  09 Sep 2005:
Vol. 309, Issue 5741, pp. 1728-1732
DOI: 10.1126/science.1117389

## Abstract

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

The ubiquity and longevity of Sanger sequencing (1) are remarkable. Analogous to semiconductors, measures of cost and production have followed exponential trends (2). High-throughput centers generate data at a speed of 20 raw bases per instrument-second and a cost of $1.00 per raw kilobase. Nonetheless, optimizations of electrophoretic methods may be reaching their limits. Meeting the challenge of the$1000 human genome requires a paradigm shift in our underlying approach to the DNA polymer (3).

Cyclic array methods, an attractive class of alternative technologies, are “multiplex” in that they leverage a single reagent volume to enzymatically manipulate thousands to millions of immobilized DNA features in parallel. Reads are built up over successive cycles of imaging-based data acquisition. Beyond this common thread, these technologies diversify in a panoply of ways: single-molecule versus multimolecule features, ordered versus disordered arrays, sequencing biochemistry, scale of miniaturization, etc. (3). Innovative proof-of-concept experiments have been reported, but are generally limited in terms of throughput, feature density, and library complexity (49). A range of practical and technical hurdles separate these test systems from competing with conventional sequencing on genomic-scale applications.

Our approach to developing a more mature alternative was guided by several considerations. (i) An integrated sequencing pipeline includes library construction, template amplification, and DNA sequencing. We therefore sought compatible protocols that multiplexed each step to an equivalent order of magnitude. (ii) As more genomes are sequenced de novo, demand will likely shift toward genomic resequencing; e.g., to look at variation between individuals. For resequencing, consensus accuracy increases in importance relative to read length because a read need only be long enough to correctly position it on a reference genome. However, a consensus accuracy of 99.99%, i.e., the Bermuda standard, would still result in hundreds of errors in a microbial genome and hundreds of thousands of errors in a mammalian genome. To avoid unacceptable numbers of false-positives, a consensus error rate of 1 × 10–6 is a more reasonable standard for which to aim. (iii) We sought to develop sequencing chemistries compatible with conventional epifluorescence imaging. Diffraction-limited optics with charge-coupled device detection achieves an excellent balance because it not only provides submicrometer resolution and high sensitivity for rapid data acquisition, but is also inexpensive and easily implemented.

Conventional shotgun libraries are constructed by cloning fragmented genomic DNA of a defined size range into an Escherichia coli vector. Sequencing reads derived from opposite ends of each fragment are termed “mate-pairs.” To avoid bottlenecks imposed by E. coli transformation, we developed a multiplexed, cell-free library construction protocol. Our strategy (Fig. 1A) uses a type IIs restriction endonuclease to bring sequences separated on the genome by ∼1 kb into proximity. Each ∼135–base pair (bp) library molecule contains two mate-paired 17- to 18-bp tags of unique genomic sequence, flanked and separated by universal sequences that are complementary to amplification or sequencing primers used in subsequent steps. The in vitro protocol (Note S1) results in a library with a complexity of ∼1 million unique, mate-paired species.

Conventionally, template amplification has been performed by bacterial colonies that must be individually picked. Polymerase colony, or polony, technologies perform multiplex amplification while maintaining spatial clustering of identical amplicons (10). These include in situ polonies (11), in situ rolling circle amplification (RCA) (12), bridge polymerase chain reaction (PCR) (13), picotiter PCR (9), and emulsion PCR (14). In emulsion PCR (ePCR), a water-in-oil emulsion permits millions of noninteracting amplifications within a milliliter-scale volume (1517). Amplification products of individual compartments are captured via inclusion of 1-μm paramagnetic beads bearing one of the PCR primers (14). Any single bead bears thousands of single-stranded copies of the same PCR product, whereas different beads bear the products of different compartmentalized PCR reactions (Fig. 1B). The beads generated by ePCR have highly desirable characteristics: high signal density, geometric uniformity, strong feature separation, and a size that is small but still resolvable by inexpensive optics.

Provided that the template molecules are sufficiently short (fig. S1), an optimized version of the ePCR protocol described by Dressman et al. (14) robustly and reproducibly amplifies our complex libraries (Note S2). In practice, ePCR yields empty, clonal, and nonclonal beads, which arise from emulsion compartments that initially have zero, one, or multiple template molecules, respectively. Increasing template concentration in an ePCR reaction boosts the fraction of amplified beads at the cost of greater nonclonality (14). To generate populations in which a high fraction of beads was both amplified and clonal, we developed a hybridization-based invitroen richment method (Fig. 1C). The protocol is capable of a fivefold enrichment of amplified beads (Note S3).

Iterative interrogation of ePCR beads (Fig. 1D) requires immobilization in a format compatible with enzymatic manipulation and epifluorescence imaging. We found that a simple acrylamide-based gel system developed for in situ polonies (6) was easily applied to ePCR beads, resulting in a ∼1.5-cm2 array of disordered, monolayered, immobilized beads (Note S4, Fig. 2A).

With few exceptions (18), sequencing biochemistries rely on the discriminatory capacities of polymerases and ligases (1, 6, 8, 1922). We evaluated a variety of sequencing protocols in our system. A four-color sequencing by ligation scheme (“degenerate ligation”) yielded the most promising results (Fig. 2, B and C). A detailed graphical description of this method is shown in fig. S7. We begin by hybridizing an “anchor primer” to one of four positions (immediately 5′ or 3′ to one of the two tags). We then perform an enyzmatic ligation reaction of the anchor primer to a population of degenerate nonamers that are labeled with fluorescent dyes. At any given cycle, the population of nonamers that is used is structured such that the identity of one of its positions is correlated with the identity of the fluorophore attached to that nonamer. To the extent that the ligase discriminates for complementarity at that queried position, the fluorescent signal allows us to infer the identity of that base (Fig. 2, B and C). After performing the ligation and four-color imaging, the anchor primer:nonamer complexes are stripped and a new cycle is begun. With T4 DNA ligase, we can obtain accurate sequence when the query position is as far as six bases from the ligation junction while ligating in the 5′→3′ direction, and seven bases from the ligation junction in the 3′→5′ direction. This allows us to access 13 bp per tag (a hexamer and heptamer separated by a 4- to 5-bp gap) and 26 bp per amplicon (2 tags × 13 bp) (fig. S7).

Although the sequencing method presented here can be performed manually, we benefited from fully automating the procedure (fig. S3). Our integrated liquid-handling and microscopy setup can be replicated with off-the-shelf components at a cost of about 140,000. A detailed description of instrumentation and software is provided in Notes S5 and S7. As a genomic-scale challenge, we sought a microbial genome that was expected, relative to a reference sequence, to contain a modest number of both expected and unexpected differences. We selected a derivative of E. coli MG1655, engineered for deficiencies in tryptophan biosynthesis and evolved for ∼200 generations under conditions of syntrophic symbiosis via coculture with a tyrosine biosynthesis–deficient strain (23). Specific phenotypes emerged during the laboratory evolution, leading to the expectation of genetic changes in addition to intentionally engineered differences. An in vitro mate-paired library was constructed from genomic DNA derived from a single clone of the evolved $Math$ strain. To sequence this library, we performed successive instrument runs with progressively higher bead densities. In an experiment ultimately yielding 30.1 Mb of sequence, 26 cycles of sequencing were performed on an array containing amplified, enriched ePCR beads. At each cycle, data were acquired for four wavelengths at 20 × optical magnification by rastering across each of 516 fields of view on the array (Fig. 1D). A detailed description of the structure of each sequencing cycle is provided in Note S6. In total, 54,696 images (14 bit, 1000 × 1000) were collected. Cycle times averaged 135 min per base (∼90 min for reactions and ∼45 min for imaging), for a total of ∼60 hours per instrument run. Image processing and base calling algorithms are detailed in Note S7. In brief, all images taken at a given raster position were aligned. Two additional image sets were acquired: brightfield images to robustly identify bead locations (Fig. 2A) and fluorescent primer images to identify amplified beads. Our algorithms detected 14 million objects within the set of brightfield images. On the basis of size, fluorescence, and overall signal coherence over the course of the sequencing run, we determined 1.6 million to be well-amplified, clonal beads (∼11%). For each cycle, mean intensities for amplified beads were extracted and normalized to a 4D unit vector (Fig. 2, B and C). The Euclidean distance of the unit vector for a given raw base call to the median centroid of the nearest cluster serves as a natural metric of the quality of that call. The reference genome consisted of the E. coli MG1655 genome (GenBank accession code U00096.2) appended with sequences corresponding to the cat gene and the lambda Red prophage, which had been engineered into the sequenced strain to replace the trp and bio operons, respectively. To systematically assess our power to detect single-base substitutions, we introduced a set of 100 random single-nucleotide changes into the reference sequence at randomly selected positions (“mock SNCs”) (Table 1). Table 1. Genome Coverage and SNC prediction. Bases with consistent consensus coverage were used to make mutation predictions. To assess power, the outcome of consensus calling for the mock SNC positions with various levels of coverage was determined. Data from two independent sets of mock SNCs are shown. “86 of 87,” for example, means that 87 of the 100 mock SNCs were present in the sequence that was covered with 1× or more reads, and 86 of these were called correctly. Coverage Percent of genome Correctly called mock substitutions 1× or greater 91.4% 86 of 87 88 of 90 2× or greater 83.3% 78 of 78 75 of 76 3× or greater 74.9% 67 of 67 68 of 68 4× or greater 66.9% 58 of 58 62 of 62 An algorithm was developed to place the discontinuous reads onto the reference sequence (Note S7). The matching criteria required the paired tags to be appropriately oriented and located within 700 to 1200 bp of one another, allowing for substitutions if exact matches were not found. Of the 1.6 million reads, we were able to confidently place ∼1.16 million (∼72%) to specific locations on the reference genome, resulting in ∼30.1 million bases of resequencing data at a median raw accuracy of 99.7%. At this stage of the analysis, the data were combined with reads from a previous instrument run that contributed an additional ∼18.1 million bases of equivalent quality (Fig. 2D). In this latter experiment, ∼1.8 million reads were generated from ∼7.6 million objects (∼24%), of which ∼0.8 million were confidently placed (∼40%). High-confidence consensus calls were determined for 70.5% of the E. coli genome for which sufficient and consistent coverage was available (3,289,465 bp; generally positions with ∼4× or greater coverage). There were six positions within this set that did not agree with the reference sequence, and thus were targeted for confirmation by Sanger sequencing. All six were correct, although in one case we detected the edge of an 8-bp deletion rather than a substitution (Table 2). Three of these six mutations represent heterogeneities in lambda Red or MG1655, or errors in the reference sequence; three were only present in the evolved variant (Table 2). Of the 100 mock SNCs, 53 were at positions called with high confidence. All of these were correctly called as substitutions of the expected nucleotide (59 of 59 on a second set of mock SNCs). The absence of substitution errors in ∼3.3 Mb of reference sequence positions called with high confidence suggests that we are achieving consensus accuracies sufficient for resequencing applications. Percentage of the genome covered and mock SNC discovery at various levels of coverage are shown in Table 1. Table 2. Polymorphism discovery. Predictions for mutated positions were tested and verified as correct by Sanger sequencing. We found three mutations unique to the evolved strain—two in ompF, a porin, and one in lrp, a global regulator. Position Type Gene Context Confirmation Comments 986,328 T → G ompF -10 region Yes Evolved strain only 931,955 8-bp deletion lrp Frameshift Yes Evolved strain only 985,791 T → G ompF Glu → Ala Yes Evolved strain only 1,976,527 - 1,977,302 776-bp deletion flhD Promoter Yes MG1655 heterogeneity 3,957,960 C → T ppiC 5′ UTR Yes MG1655 heterogeneity λ-red, 3274 T → C ORF61 Lys → Gly Yes λ-red heterogeneity λ-red, 9846 T → C cl Glu → Glu Yes λ-red heterogeneity Despite 10× coverage in terms of raw base pairs, only ∼91.4% of the genome had at least 1× coverage (fig. S4). Substantial fluctuations in coverage were observed owing to the stochasticity of the RCA step of library construction. We are currently generating libraries that are more complex and more evenly distributed. A Gaussoid distribution of distances between mate-paired tags was observed, consistent with the size selection during library construction (Fig. 3, A and B). Notably, the helical pitch of DNA (∼10.6 bp per turn) is evident in the local statistics of ∼1 million circularization events (Fig. 3B). As a function of the number of bases sequenced, we generated over an order of magnitude more mate-pairing data points than an equivalent amount of conventional sequencing. To detect genomic rearrangements, we mined the unplaced mate-pairs for consistent links between genomic regions that did not fall within the expected distance constraints. In addition to detecting the expected replacements of the trp and bio operons with cat and lambda Red prophage (Fig. 3D), we detected and confirmed the absence of a 776-bp IS1 transposon (Fig. 3C), a previously described heterogeneity in MG1655 strains (24). We also detected and confirmed a ∼1.8-kb region that was heterogeneously inverted in the genomic DNA used to construct the library (Fig. 3E), owing to activity of pin on the invertible P region (25). We observe error rates of ∼0.001 for the better half of our raw base calls (Fig. 2D). Although high consensus accuracies are still achieved with relatively low coverage, our best raw accuracies are notably one to two orders of magnitude less accurate than most raw bases in a conventional Sanger sequencing trace. The PCR amplifications before sequencing are potentially introducing errors at a rate that imposes a ceiling on the accuracies achievable by the sequencing method itself. One potential solution is to create a library directly from the genomic material to be sequenced, such that the library molecules are linear RCA amplicons. Such concatemers, where each copy is independently derived from the original template, would theoretically provide a form of error correction during ePCR. Our algorithms were focused on detection of point substitutions and rearrangements. Increasing read lengths, currently totaling only 26 bp per amplicon, will be critical to detecting a wider spectrum of mutation. A higher fidelity ligase (20) or sequential nonamer ligations (20, 21) may enable completion of each 17- to 18-bp tag. Eco P15 I, which generates ∼27-bp tags, would allow even longer read lengths while retaining the same mate-pairing scheme (26). We estimate a cost of0.11 per raw kilobase of sequence generated (Note S8), roughly one-ninth as much as the best costs for electrophoretic sequencing. Raw data in all sequencing methods are generally combined to form a consensus. Even though costs are generally defined in terms of raw bases, the critical metric to compare technologies is consensus accuracy for a given cost. There is thus a need to devise appropriate cost metrics for specific levels of consensus accuracy.

If library construction costs are not included, the estimated cost drops to \$0.08 per raw kilobase. Higher densities of amplified beads are expected to boost the number of bases sequenced per experiment. While imaging, data were collected at a rate of ∼400 bp/s. Although enzymatic steps slowed our overall throughput to ∼140 bp/s, a dual flowcell instrument (such that the microscope is always imaging) will allow us to achieve continuous data acquisition. Enzymatic reagents, which dominate our cost equation, can be produced in-house at a fraction of the commercial price.

We demonstrate low costs of sequencing, mate-paired reads, high multiplicities, and high consensus accuracies. These enable applications including BAC (bacterial artificial chromosome) and bacterial genome resequencing, as well as SAGE (serial analysis of gene expression) tag and barcode sequencing. Simulations suggest that the current mate-paired libraries are compatible with human genome resequencing, provided that the read length can be increased to cover the full 17- to 18-bp tag (fig. S5).

What are the limits of this approach? As many as 1 billion 1-μm beads can potentially be fit in the area of a standard microscope slide (fig. S6). We achieve raw data acquisition rates of ∼400 bp/s, more than an order of magnitude faster than conventional sequencing. From another point of view, we collected ∼786 gigabits of image data from which we gleaned only ∼60 megabits of sequence. This sparsity—one useful bit of information per 10,000 bits collected—is a ripe avenue for improvement. The natural limit of this direction is single-pixel sequencing, in which the commonplace analogy between bytes and bases will be at its most manifest.

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