SPECIAL FEATURE
MEETING REPORT
Report on the Sequencing by Hybridization Workshop C. R. CANTOR, A. MIRZABEKOV, AND E. SOUTHERN Overview Few biologists have heard the acronym SBH (sequencing by hybridization), which describes a set of related technologies that may revolutionize the practice of DNA sequencing. The potential of this general approach and the current state of its art were assessed at a recent workshop organized by HUGO (the Human Genome Organization) and supported by the U.S. Department of Energy (DOE), the Wellcome Trust (UK), and the Human Genome Project of the Russian Federation. This workshop took place in Moscow on November 19-20,199l. It seems appropriate that this first international workshop on SBH was held in eastern Europe, since two of the four independent original initiatives in this approach began there; the meeting participants were able to view firsthand some of the samples and apparatus constructed for SBH in Moscow and meet many of the younger scientists involved in these endeavors. The workshop comprised 2 days of oral presentations and intense discussion. The venue was the Englehardt Institute of Molecular Biology in Moscow. The 44 participants came from four countries: Russian Federation (19), United States (14), United Kingdom (9), and Sweden (2), representing government and university research laboratories, and several large and small companies. Although the attendees were drawn mostly from active practitioners in this embryonic field, the overall attitude was a healthy skepticism and a genuine realization of the difficulties inherent in attempting to develop a radically different approach to one of the most commonly used tools in biology. Like traditional DNA sequencing, SBH raises concerns about sample preparation and data analysis and integration, in addition to the obvious issues involved in reading raw sequence. There are two basic formats for SBH. In Format 1, a single oligonucleotide probe is used to examine an array of immobilized samples. This array can be of any size, depending on the nature of the samples. In Format 2, a single sample is hybridized to a chip containing an array of oligonucleotides. A general consensus of the meeting was that one or both of these SBH formats are likely to work as practical sequencing tools in the near future. For example, a chip for sequencing hundreds to thousands of nucleotides might cost from a few dollars to tens of dollars when made by mass production. The sequencing procedure for using such a chip could easily be automated, and the speed of such sequencing on an automated instrument could approach millions of bases per day. A number of obstacles remain to be solved. Foremost among them is finding ways to compensate for the base composition and sequence dependence of the stability of short oligonucleo-
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tide duplexes. To overcome these obstacles, a major expansion in SBH activities is needed, because such data can be obtained only by the highly parallel kinds of hybridization studies that will lie at the heart of future implementations of SBH. Progress to date has been impressive, and calculations of future potential remain quite optimistic. Thus it would appear foolish to delay efforts to construct the arrays of samples needed to implement such parallel studies. The field is not yet at a stage where a single approach can be judged most promising. A number of efforts should proceed simultaneously, varying key ingredients such as the surface to which samples are attached, the kinds of samples attached, the hybridization conditions, and the manner in which hybridization is detected. There was a strong consensus that the field is at a stage where it could benefit enormously from extensive international cooperation. For example, much of the current development of SBH procedures is taking place in Russia. Considering the present economic difficulties in that country, it is essential to take measures to ensure the effective involvement of Russian scientists in international cooperative efforts. The various implementations of SBH share many needs, including samples, detectors, and data analysis algorithms. The relative efficacy of the various approaches to these problems will be much easier to assess if different research groups have access to each other’s samples, raw data, and analyses. Indeed, it is quite likely that the method that ultimately proves most efficient and reliable could be a fusion of a number of different approaches. For this reason, a major recommendation by the meeting participants was to put in place the mechanisms for promoting an unusual degree of long-range cooperation. These included direct access to raw data generated by each of the laboratories involved, free exchange of software, facilitation of scientific visits, and an annual workshop to assess progress and promote shared technology. In addition, as work proceeds, it should be possible to share or exchange samples such as large numbers of oligonucleotides or arrays in such a way as to reduce the overall cost of development efforts and facilitate comparison among various specific SBH implementations. The degree of cooperation desirable in SBH research is driven by a feature of this field that is unusual in most biological efforts, but is rather common in physics. The initial cost of full-scale implementation of SBH will be quite a high percentage of typical overall costs in using SBH to achieve stated goals of large-scale determination of DNA sequences. Thus it is important to carefully integrate and continually assess SBH efforts during this expensive development stage. The overall scheme for SBH development is fairly similar to the construction of a new tool for high-energy physics. Technical expertise from many different disciplines must be combined, a large expenditure must be committed long before the success of the project (in terms of new fundamental discoveries, for physics; and successful large-scale DNA sequencing, for biology) can be demonstrated. Indeed, a realistic appraisal of SBH key parameters, such as DNA sequence throughput, sequence accuracy, and ability to integrate sequence fragments, will probably be
0888-7543/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
SPECIAL FEATURE possible only several years from now. However, the promise of this new methodology is great and the likelihood of its success is very high, so that the gamble to proceed on a large scale must now be taken. Certain key choices about approaches likely to succeed can be distilled from some of the detailed reports of specific sessions of the meeting, but many variables remain to be explored in parallel. In a workshop that ricocheted from very simple pilot feasibility tests to grand future schemes, it is difficult to cull only a few highlights, but here are several: SBH can produce DNA sequence de nouo, as demonstrated in a pilot test with several related but unknown sequences by Crkvenjakov and Drmanac. Problems of discrimination between correctly paired and end mismatched oligonucleotide pairs can be reduced very substantially if the mismatch occurs next to an adjacent DNA duplex. This procedure has been termed oligonucleotide stacking hybridization (Fig. 1). The mode of surface immobilization and the nature of the surface used have major effects on SBH, and these need extensive further study. The very nature of some of the potential ambiguities in SBH mandates that data analysis algorithms produce statistical estimates of the likelihood of particular sequences being consistent with available data. Thus SBH may actually put a statistical view of DNA sequencing results in place before this is employed in more conventional DNA sequencing. The costs of SBH development were not fully assessed at the meeting. Indeed, it is by no means clear yet that the full potential of SBH has been conceived. The power of SBH for sequence comparison and for clinical diagnostics seems unquestioned. Thus there is a good rationale for funding a number of parallel efforts in all phases of SBH. The time for coalescence into a more unified approach is several years hence. Premature specialization could be a costly error. For example, most participants appear to favor SBH Format 2 for DNA sequencing. Yet for mapping by SBH, strong arguments can be made for SBH Format 1. Such mapping appears to be making very good progress as judged by Lehrach’s work on Schizosaccharomyces pombe, and it certainly deserves further elaboration. Similarly, the relative merits of hybridization detection by radioisotope decay, fluorescence, dielectric properties, or mass spectrometry remain to be proven. The Moscow workshop on SBH was unusually stimulating. It may be a while before this new approach is generally accepted, but the participants of the meeting, with few if any exceptions, left firmly committed to the endeavor. Principles and Practice of Oligonucleotide Hybridization J. Wetmur discussed the fact that hybridization of oligonucleotides depends either on the concentration of the sequence in excess and the melting temperature, T,, an equilibrium property, or on the time of washing and the dissociation temperature, Td, a kinetic property (the temperature at which half of a preformed duplex is released in a specified time). Because the temperature dependence on the rate of duplex formation is 5’ 3’
-
+
+ FIGURE
5’ 3’
very slight, both T, and Td are dominated by the activation energy for helix dissociation. Thus T,,, and Td depend in the same manner on DNA sequence and the effect of mismatched sequences on hybridization. The ability to calculate these characteristic temperatures for short oligonucleotides using nearest-neighbor data is rather good, but is still limited by incomplete data on the sequence-dependent effects of dangling ends and mismatches, especially those producing stable non-Watson-Crick structures. Formats envisioned for SBH include (1) hybridization of single oligonucleotides to arrays of immobilized cloned DNA and (2) hybridization of DNA from a single clone to arrays of oligonucleotides. A review of the thermodynamics and kinetics of oligonucleotide hybridization in solution reveals that these processes are the same, provided that the length of the cloned DNA in solution is reduced to the effective length of the cloned DNA on a solid support. Reducing the size of the strands would also decrease possible confounding effects of secondary structure. A variation on Format 2 involves release of hybridized cloned DNA fragments from oligonucleotides immobilized in a gel matrix. In this case, the reaction is governed by a pre-equilibrium (T,,,) and a rate-determining step involving diffusion through the gel. A. Mirzabekov indidated that one of the difficulties with SBH is duplicated short sequences in the cloned DNA. This difficulty may be reduced for longer DNAs either by using longer immobilized oligonucleotides or by using end-to-end continuous stacking of adjacent oligonucleotides hybridized for the DNA target. This increases the Td of the duplex formed and is equivalent to working with a much more complex matrix. For example, a matrix of immobilized octanucleotides used together with a preselected solution mixture of pentanucleotides can produce the same results as a matrix with immobilized tridecanucleotides. R. Crkvenjakov and R. Drmanac, using Format 1, and Mirzabekov, using Format 2 in a gel, demonstrated in dot blots and in arrays of cloned DNA the ability to distinguish hybridization of very short oligonuleotides to complementary sequences from hybridization to mismatched sequences or from nonspecific binding to background. J. Brown demonstrated the properties of synthetic nucleotides for inclusion in oligonucleotides that can substitute either for both purine (A and G) or for both pyrimidine (T and C) nucleotides. These molecules may prove useful for the practical synthesis of partially degenerate longer oligonucleotides for use in either format of SBH. (See discussion of gapped probes.) Oligonucleotide
Synthesis
K. Beattie described the multiple DNA synthesis technology that was developed in his laboratory at Baylor College of Medicine and subsequently commercialized by Genosys Biotechnologies, Inc. The segmented synthesis approach taken by Genosys employs stacks of porous Teflon wafers, each comprising a different reaction chamber containing derivatized support material, to simultaneously add a given base to all sequences within the stack. Following each reaction cycle (phosphoramidite method of synthesis), the wafers are sorted into different stacks to permit synthesis of a different se-
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SPECIAL FEATURE quence within each wafer. Genosys’ current prototype instrument, automated in all aspects except for wafer sorting, is capable of synthesizing 100 oligonucleotides simultaneously with a cycle time of 6 min, including computer-directed manual sorting. The reagent cost at 0.2.hmol scale of synthesis is currently $0.25-$0.50 per base, and the product yield and quality are the same as for conventional automated DNA synthesizers. Automation of the Genosys machine is being continued with SBIR grant support from NIH. Beattie pointed out that if 10 of these Genosys machines were put to work synthesizing oligonucleotide libraries, the entire library of 65,536 octamers could be prepared in less than 2 weeks, and the entire library of 1,048,576 decamers could be synthesized in about 200 days. Efficient storage and retrieval of large oligonucleotides libraries are challenges that Genosys will address in a DOE-funded small business incentive research (SBIR) project. Beattie described some feasibilty studies that were performed in a collaboration between Baylor College of Medicine and Houston Advanced Research Center and aimed at fabrication of DNA chips containing hundreds to thousands of surface-bound probes acting as “genosensor” elements. Glass surfaces were derivatized by epoxysilane, then reacted with 5’aminoalkyl nonamer probes. The probes were shown to bind specifically at their Y-amino termini, spaced approximately 50 A apart on the surface, and when hybridized with 5’-32P-labeled 36-mer target DNA samples, gave good discrimination against internal mismatches. The HARC/BCM team, in collaboration with microfabrication engineers at MIT Lincoln Laboratory and a microrobotics collaborator, plans to produce miniaturized genosensors containing up to several thousand probes spaced 50-100 pm apart within a l-cm’ area. Beattie’s laboratory at HARC will be developing genosensor applications for genomic mapping and mutation detection. S. Fodor outlined the relative advantages and disadvantages of the off-chip and on-chip strategies for preparation of DNA probe arrays and described the approach being pursued by Affymax Research Corp., which utilizes addressable laser-activated photodeprotection in the chemical synthesis of oligonucleotides (or peptides) directly on a glass surface. Affymax scientists have recently developed new phosphoramidite derivatives capable of highly efficient light-activated detritylation and will now be evaluating these reagents for producing miniaturized DNA chips, including an octamer chip within a 1-in.2 area. E. Southern of Oxford University described his laboratory’s experience with SBH Format 2. They have produced DNA probe arrays by an on-chip strategy that employs a stable linkage comprising a 20.atom aliphatic chain attached to a glass surface with a primary hydroxyl group at the other end which is used to initiate synthesis of oligonucleotides in situ. Oligonucleotide arrays were built by directing the base additions to channels created by barriers between plates; alternate bases are added through channels placed at right angles to the previous addition, and finer spacing is used as the length of the oligonucleotides and the complexity of the set increase. Using this approach, a complete array of 4” s-mers is achieved in s cycles, e.g., 65,536 octamers in eight cycles. Thus far the Oxford group has made arrays of 4096 oligonucleotides on plates of 20 X 20 cm.
SBH Results:
Format
1
In the session on SBH Format 1 in which complex target DNAs are on a surface and the oligonucleotide is in solution, results were shown by five speakers from two groups: ICRF (headed by H. Lehrach) and Argonne National Lab (headed by Drmanac/Crkvenjakov). H. Lehrach presented a mapping strategy for linkup of cosmids via oligonucleotide fingerprint matching and announced imminent ordering of the S. pombe genome in cosmids. The reference library concept as an integral means of organizing information gathering in mapping and sequencing via shared filters with arrayed clones was presented, and the major role of oligonucleotide hybridization in this inquiry emphasized. New technology development in London is in the area of robotic formation of dense filter arrays from microtiter plate samples with more than the standard 96 wells. Pilot experiments with oligomers substituted with modified bases to enhance binding were presented by J. Hoheisel. A full commitment to a project for providing signatures for partial sequences of clones in cDNA libraries by SBH was announced. Crkvenjakov showed results of two model sequencing experiments, one starting with the known sequence and the other with the sequence not known to the investigators. In the blind test, the Argonne group determined in parallel the sequence of 111/116-bp stretches in three related inserts using a supplied probe set guaranteed to contain at least one subset that defined one of the sequences by the maximal overlap of its members. The results obtained with 164 probes uniquely defined the three sequences by positive hybridization of more than five probes per basepair on average. When the test results were evaluated, it was found that no wrong bases were called in 343 bp of hybridization-determined sequence. The maximal data collection error rate was 3.25%, and the real rate is probably lower. Drmanac presented results on the development of technological components of a hybridization data production line based on Ml3 clone libraries. Parallel clone placement, growth, sample preparation, and robotic spotting on filters in dense arrays based on microtiter plate format were shown to work at performance levels that allow collection of up to 10 million cloneprobe hybridization data per day. For example, 13,824 dots were made on an 8 X 12-cm filter by offset printing of samples from 144 microtiter plates. This extrapolates to a measurement capacity of hybridization signals from over 150,000 dots/ 30 min scanning of a single screen using a Phosphorimager instrument (Molecular Dynamics, Sunnyvale, CA). Lehrach and Crkvenjakov summarized the results on Format 1 SBH by concluding that the time is ripe for large volume data collection experiments both in mapping and in partial to complete sequencing. The potential for dissemination and immediate applicability of the technical developments in this area in a wide variety of genome-related problems was stressed by pointing out that SBH is the natural outgrowth of familiar and widely used methods of molecular biology. Probe and Sample SBH dominantly depends analysis. Six talks presented
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computational section of the
SPECIAL FEATURE Moscow SBH meeting are a healthy sign of progress. The main problem of SBH is frequent occurrence of branching points, especially in data containing errors and in some sequences comprising short, partly degenerate tandemly repeated oligonucleotide sequences. The informatics solutions presented could reduce the required numbers of scored probe-clone hybridization data per basepair of genome sequence by up to 1000 times. One solution described by W. Bains is gapped probes (B,N,B, or B,I,,,B,, where B, and B, are unique base sequences, N,,, is a stretch of fully degenerate bases, and 1, is a stretch of m inosines). His results, and those of Yu. Lysov, P. Pevzner, and R. Lipshutz, demonstrate that gapped probes will have 10 times more power than ordinary probes. Lysov showed calculations revealing a high efficiency of solving branching points using the method of continuous stacking hybridization described earlier for a few additional hybridization cycles. In addition to modified probe design, higher efficiency can be obtained by mathematical solutions that operate with relative intensities of hybridization signals instead of assuming all or none hybridization. The efficiency of sequence reconstruction depends on a correct definition of the error function for the data. J. Elder demonstrated successful sequence reconstruction with low discrimination but highly redundant hybridization data. For sequencing complex genomes with extensive polymorphisms, SBH appears to be especially powerful. Drmanac presented data from a few small-scale simulation experiments, demonstrating efficient use of overlapped DNA fragments and comparison of similar sequences for solving branching points. As a consequence, 10 million to 1-4-kb-long genomic clones hybridized with 2000 6- to 8-mer probes may be sufficient for almost complete sequence reconstruction of the human genome. Drmanac’s view of the next step in SBH informatics is developing efficient software for unifying mathematical and heuristic solutions and optimizing the parameters in comprehensive simulation experiments on the megabase range. Drmanac concluded that appropriate combinations of conventional gel sequencing and SBH can be more efficient than any one method alone for genome sequencing. What is more important, genome sequencing with the two methods combined can be implemented immediately using SBH Format 1. SBH Results:
Format
2
Format 2 is based on hybridization of DNA to a matrix of immobilized overlapping oligonucleotides, identification of oligonucleotides that upon hybridization form perfect duplexes with DNA, and reconstitution of DNA sequence by searching for hybridized oligonucleotides that overlap with each other. U. Maskos described experiments with complete arrays (5 x 5 cm) of octapurine sequences. Hybridization with a complete set of 256 octapyrimidines was used to find conditions that minimize the effects of base composition on duplex yield. Tetramethylammonium chloride was found to remove much of the effect, but some sequence-dependent differences remain. The same data were used to calibrate the array in model sequencing and mutation analyses, using synthetic pyrimidine 20-mers. The algorithms developed by Elder, described below, produced the correct sequences. In a novel approach to the
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detection of sequence differences, two digital images produced by hybridizing two related sequences to the same array were subtracted. The sequence difference was derived from the difference data. Maskos also described small arrays (90 oligonucleotides) for optimizing the choice of allele-specific oligonucleotides (ASOs) used in diagnosis of genetic diseases. Using the welltried example of the sickle cell mutation in the P-globin gene, he showed excellent discrimination between wildtype and mutant alleles. Significantly, probes 110 nucleotides long, made from PCR products, behaved as well in hybridizations as synthetic 19.mers. An advantage of the glass plate technology is that ASOs can be laid down in stripes so that multiple probes can be laid across them in orthogonal stripes to introduce parallelism into the analysis. For complete sequencing, it could be necessary to have a matrix of all possible oligomers, for example, all 65,536 octanucleotides. Mirzabekov estimated that the method could be realized in 2-5 years depending on available resources and predicted that it will provide a fast and inexpensive sequencing procedure that could be easily automated and used in many laboratories for sequencing millions of bases per day. Partial sequencing or comparison of different sequences would need only partial matrices. This can be developed in 1-3 years. Diagnosis of genetic diseases and changes in the sequence of genes with known st.ructure would need a matrix of only tens to thousands of immobilized oligonucleotides complementary to the whole gene sequence or a part of it. This could be developed in a year, if based on PCR DNA amplification. Analysis could be performed rapidly and inexpensively. Mirzabekov demonstrated that discrimination of perfect duplexes from imperfect ones containing internal mismatches and often also terminal mismatches can be carried out by measuring hybridization intensities. The Moscow group has made progress in the manufacturing of a sequencing chip, a micromatrix of immobilized oligonucleotides. The use of gels fixed on a glass plate as a support for immobilization has been patented. Technologies have also been developed for making 30 X 30 to 100 X 100 ym gel squares that are lo-30 ,um thick, for applying oligonucleotide solutions to these. Chemistry for quantitative and fast immobilization has been developed. Modifications of these and other novel technologies are under development for commercial production of chips. At present, the Moscow group can produce chips containing tens of immobilized oligonucleotides. In the near future, chips with hundreds of oligonucleotides are expected. By using modified microelectronic technologies, it should be possible to produce thousands of chips containing hundreds of thousands of immobilized oligonucleotides at a cost of $1-50 each. The Moscow group is using fluorescent tags for DNA labeling and quantitative detection of hybridization. Fluorescence appears to be better t,han radioactive measurements in resolution, speed, safety, and other characteristics. Fluorescence can be used with the microchips described above. Prototype equipment for quantitative fluorescent measuring of hybridization has been constructed. It consists of a fluorescent microscope, a CCD camera, a thermostated chamber for hybridization, and a computer with relevant programs for image analysis and se-
SPECIAL FEATURE quence reconstruction operations. The cost of the fully automated equipment could be less than $50,000. Some major difficulties remain to be overcome. 1. Complexity ofthe chip: The longer the immobilized oligonucleotides, the higher the efficiency of sequencing, but the complexity of the chip needed grows exponentially. A chip with 65,536 immobilized octanucleotides would enable sequencing DNAs about 200-400 bases long. This matrix offers a compromise between efficiency and complexity. Mirzabekov suggested a procedure consisting of several rounds of hybridization of DNA with the same octanucleotide matrix in the presence of different short oligonucleotides (preselected by computer), such as pentanucleotides, to reach the efficiencies typical of a matrix with immobilized 13.mers. This will enable efficient sequencing of DNAs 3000-5000 nucleotides long, in a few minutes. The method is based on continuous stacking hybridization illustrated earlier. 2. Different stability of A-T and G-C basepairs in DNA duplexes: Generally, hybridization measurements need to be carried out at different temperatures since duplexes with different A-T basepair contents have different thermostabilities. By using gel-immobilized oligonucleotides, the Moscow group found that the thermal stability of duplexes depends on the concentration of immobilized oligonucleotides. However, at a properly chosen concentration of immobilized oligonucleot.ides. it becomes possible to build up a normalized matrix where all oligonucleotides form duplexes with similar or even equal thermostability despite differences in their A-T contents. 3. DNA size and its secondary structure: Long DNA fragments can form secondary structures that interfere with hybridization. Therefore, fragmentation of DNA to rather short fragments is necessary, preferentially with a narrow size distribution. Several approaches for DNA fragmentation to specific size have been developed in MOSCOW. Informatics The highlights of the informatics section of the SBH conference in Moscow were new ideas for modeling hybridization, fragment reconstruction, and sequencing chip design. J. Elder from Southern’s group at Oxford presented a linear model for going from sequence to detected SBH signal. The model is given by D = R(F) + s*e, where D is the observed data. R is a linear response function, F is the multiplicity of each oligonucleotide in a candidate fragment, s is the standard deviation of the noise, and e is a multidimensional normal variable. If D, F. and e are vectors, then R is a matrix and s is a scalar. Elder went on to describe an implementation of the model that derives R and s from the data using maximum likelihood and maximum entropy considerations. An example using data from Southern’s chip was shown. This general model and the associated parameter estimation techniques provide a solid basis for further development of the hybridization models. R. Hagstrom from Argonne National Laboratory introduced a new methodology for fragment reconstruction. Specifically, this new methodology takes the hybridization data and yields a directed Eulerian multigraph. (This is a series of points [vertices] connected by multiple paths with directions assigned to
each path, such that all the paths can be traced without any duplication. An example is a solution to the classic mathematical problem: the seven bridges of Konigsberg.) Possible fragments correspond to Eulerian paths in the graph. Suppose G is an Eulerian multigraph, and D is the hybridization data; then let UD 1G) be the likelihood that any fragment G corresponding to G yields data D. Then, given hybridization D, the new algorithm finds a graph G that maximizes L(D / C). A specific implementation of the algorithm for linear response function and normal errors was presented. It was shown that this algorithm has polynomial complexity. This new algorithm significantly advances work on the problem of fragment reconstruction. It subsumes the previous work of Pevzner and Lipshutz and is flexible enough to allow significant modification of the definition of L(D 1G). This will allow adjustment of the method as our understanding of the hybridization process improves. Bains, Lipshutz, and Lysov presented work on the selection and design of a highly efficient sequencing chip. The concept of a probe was extended to be a set of oligonucleotides colocated on a chip or applied to all targets simultaneously. A chip is then a collection of such sets. For a fixed number of probes, the efficiency of a chip can be measured by the maximum length of a fragment such that the probability of branching on reconstruction is less than a fixed probability. Bains described simple probes with internal gaps. Lysov described families of such probes. Lipshutz described three very general sets of probes called gapped (described earlier), alternating (for example, ANGNGNC where N is any base), and binary (for example, YRRYYRY, where Y is pyrimidine and R is purine). Efhciencies for the binary family were described. It was shown that for a fixed capacity the efficiency varies with the square root of the probability of branching for binary chips while it varies linearly in the case of standard probes. These results point the way to being able to unambiguously sequence longer fragments using the same or a smaller number of probes. Bains also presented an initial analysis of the probability of repeated 8- tuples as a function of their separation. This analysis was performed using the human hemoglobin locus. The function is damped and periodic. This is a surprising result and is quite different from the result using random sequence data. This result will have significant impact on predicting the efficiency of SBH. In particular, future SBH simulations must be performed using actual sequence data. Lipshutz present updated work on maximum likelihood fragment reconstruction. Simulated results based upon the improved model were shown. Drmanac presented his earlier work on heuristics for fragment reconstruction.
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Hybridization
Detection
Detection techniques presented ranged from low cost/moderate sensitivity to high cost/high sensitivity. Specifically, four detection schemes were presented. The first technique was conventional radioactive labeling, which is a proven technology yet not amenable to miniaturization due to the l-mm resolution limit. Next, the optical approach that relies on fluerescent tags serving as markers for hybridized DNA was presented. Early results indicate at least a 3:l discrimination factor for hybridization detection. Such approach is amenable to
SPECIAL FEATURE miniaturization, yet requires costly laser optical equipment. The third approach presented was derived from mass spectroscopy. This approach entails vaporizing the surface of a stable isotope-labeled sample with a probe beam scanned in a 2-dimensional pattern and subsequently detecting the composition of the sample with a tuned laser. The discrimination factor for such an approach exceeds lOO:l, with an incredible sensitivity of a few parts in a trillion, basically detecting a few atoms per isotope. The trade-off for such sensitivity was seen to be the cost in supplying the powerful laser equipment. The last technique described is a direct detection approach, in which no tags are required to mark the samples for improved discrimination. A microelectronic detection technique
that relies on a change in the dielectric properties in a localized DNA probe test well was introduced, constructed using standard microfabrication techniques. The microelectronic approach provided a 1O:l discrimination factor in preliminary hybridization experiments. Moreover, the method has the potential of providing an extremely low-cost solution to hybridization detection, since the accompanying instrumentation can be placed directly on the microchip with the hybridization array. Overall, the potentially viable hybridization detection techniques discussed were summarized in terms of maturity, speed, cost, resolution, preparation, training, hazard, and instrumentation.
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MEETING REPORT
Report on the Sequencing by Hybridization Workshop C. R. CANTOR, A. MIRZABEKOV, AND E. SOUTHERN Overview Few biologists have heard the acronym SBH (sequencing by hybridization), which describes a set of related technologies that may revolutionize the practice of DNA sequencing. The potential of this general approach and the current state of its art were assessed at a recent workshop organized by HUGO (the Human Genome Organization) and supported by the U.S. Department of Energy (DOE), the Wellcome Trust (UK), and the Human Genome Project of the Russian Federation. This workshop took place in Moscow on November 19-20,199l. It seems appropriate that this first international workshop on SBH was held in eastern Europe, since two of the four independent original initiatives in this approach began there; the meeting participants were able to view firsthand some of the samples and apparatus constructed for SBH in Moscow and meet many of the younger scientists involved in these endeavors. The workshop comprised 2 days of oral presentations and intense discussion. The venue was the Englehardt Institute of Molecular Biology in Moscow. The 44 participants came from four countries: Russian Federation (19), United States (14), United Kingdom (9), and Sweden (2), representing government and university research laboratories, and several large and small companies. Although the attendees were drawn mostly from active practitioners in this embryonic field, the overall attitude was a healthy skepticism and a genuine realization of the difficulties inherent in attempting to develop a radically different approach to one of the most commonly used tools in biology. Like traditional DNA sequencing, SBH raises concerns about sample preparation and data analysis and integration, in addition to the obvious issues involved in reading raw sequence. There are two basic formats for SBH. In Format 1, a single oligonucleotide probe is used to examine an array of immobilized samples. This array can be of any size, depending on the nature of the samples. In Format 2, a single sample is hybridized to a chip containing an array of oligonucleotides. A general consensus of the meeting was that one or both of these SBH formats are likely to work as practical sequencing tools in the near future. For example, a chip for sequencing hundreds to thousands of nucleotides might cost from a few dollars to tens of dollars when made by mass production. The sequencing procedure for using such a chip could easily be automated, and the speed of such sequencing on an automated instrument could approach millions of bases per day. A number of obstacles remain to be solved. Foremost among them is finding ways to compensate for the base composition and sequence dependence of the stability of short oligonucleo-
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tide duplexes. To overcome these obstacles, a major expansion in SBH activities is needed, because such data can be obtained only by the highly parallel kinds of hybridization studies that will lie at the heart of future implementations of SBH. Progress to date has been impressive, and calculations of future potential remain quite optimistic. Thus it would appear foolish to delay efforts to construct the arrays of samples needed to implement such parallel studies. The field is not yet at a stage where a single approach can be judged most promising. A number of efforts should proceed simultaneously, varying key ingredients such as the surface to which samples are attached, the kinds of samples attached, the hybridization conditions, and the manner in which hybridization is detected. There was a strong consensus that the field is at a stage where it could benefit enormously from extensive international cooperation. For example, much of the current development of SBH procedures is taking place in Russia. Considering the present economic difficulties in that country, it is essential to take measures to ensure the effective involvement of Russian scientists in international cooperative efforts. The various implementations of SBH share many needs, including samples, detectors, and data analysis algorithms. The relative efficacy of the various approaches to these problems will be much easier to assess if different research groups have access to each other’s samples, raw data, and analyses. Indeed, it is quite likely that the method that ultimately proves most efficient and reliable could be a fusion of a number of different approaches. For this reason, a major recommendation by the meeting participants was to put in place the mechanisms for promoting an unusual degree of long-range cooperation. These included direct access to raw data generated by each of the laboratories involved, free exchange of software, facilitation of scientific visits, and an annual workshop to assess progress and promote shared technology. In addition, as work proceeds, it should be possible to share or exchange samples such as large numbers of oligonucleotides or arrays in such a way as to reduce the overall cost of development efforts and facilitate comparison among various specific SBH implementations. The degree of cooperation desirable in SBH research is driven by a feature of this field that is unusual in most biological efforts, but is rather common in physics. The initial cost of full-scale implementation of SBH will be quite a high percentage of typical overall costs in using SBH to achieve stated goals of large-scale determination of DNA sequences. Thus it is important to carefully integrate and continually assess SBH efforts during this expensive development stage. The overall scheme for SBH development is fairly similar to the construction of a new tool for high-energy physics. Technical expertise from many different disciplines must be combined, a large expenditure must be committed long before the success of the project (in terms of new fundamental discoveries, for physics; and successful large-scale DNA sequencing, for biology) can be demonstrated. Indeed, a realistic appraisal of SBH key parameters, such as DNA sequence throughput, sequence accuracy, and ability to integrate sequence fragments, will probably be
0888-7543/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
SPECIAL FEATURE possible only several years from now. However, the promise of this new methodology is great and the likelihood of its success is very high, so that the gamble to proceed on a large scale must now be taken. Certain key choices about approaches likely to succeed can be distilled from some of the detailed reports of specific sessions of the meeting, but many variables remain to be explored in parallel. In a workshop that ricocheted from very simple pilot feasibility tests to grand future schemes, it is difficult to cull only a few highlights, but here are several: SBH can produce DNA sequence de nouo, as demonstrated in a pilot test with several related but unknown sequences by Crkvenjakov and Drmanac. Problems of discrimination between correctly paired and end mismatched oligonucleotide pairs can be reduced very substantially if the mismatch occurs next to an adjacent DNA duplex. This procedure has been termed oligonucleotide stacking hybridization (Fig. 1). The mode of surface immobilization and the nature of the surface used have major effects on SBH, and these need extensive further study. The very nature of some of the potential ambiguities in SBH mandates that data analysis algorithms produce statistical estimates of the likelihood of particular sequences being consistent with available data. Thus SBH may actually put a statistical view of DNA sequencing results in place before this is employed in more conventional DNA sequencing. The costs of SBH development were not fully assessed at the meeting. Indeed, it is by no means clear yet that the full potential of SBH has been conceived. The power of SBH for sequence comparison and for clinical diagnostics seems unquestioned. Thus there is a good rationale for funding a number of parallel efforts in all phases of SBH. The time for coalescence into a more unified approach is several years hence. Premature specialization could be a costly error. For example, most participants appear to favor SBH Format 2 for DNA sequencing. Yet for mapping by SBH, strong arguments can be made for SBH Format 1. Such mapping appears to be making very good progress as judged by Lehrach’s work on Schizosaccharomyces pombe, and it certainly deserves further elaboration. Similarly, the relative merits of hybridization detection by radioisotope decay, fluorescence, dielectric properties, or mass spectrometry remain to be proven. The Moscow workshop on SBH was unusually stimulating. It may be a while before this new approach is generally accepted, but the participants of the meeting, with few if any exceptions, left firmly committed to the endeavor. Principles and Practice of Oligonucleotide Hybridization J. Wetmur discussed the fact that hybridization of oligonucleotides depends either on the concentration of the sequence in excess and the melting temperature, T,, an equilibrium property, or on the time of washing and the dissociation temperature, Td, a kinetic property (the temperature at which half of a preformed duplex is released in a specified time). Because the temperature dependence on the rate of duplex formation is 5’ 3’
-
+
+ FIGURE
5’ 3’
very slight, both T, and Td are dominated by the activation energy for helix dissociation. Thus T,,, and Td depend in the same manner on DNA sequence and the effect of mismatched sequences on hybridization. The ability to calculate these characteristic temperatures for short oligonucleotides using nearest-neighbor data is rather good, but is still limited by incomplete data on the sequence-dependent effects of dangling ends and mismatches, especially those producing stable non-Watson-Crick structures. Formats envisioned for SBH include (1) hybridization of single oligonucleotides to arrays of immobilized cloned DNA and (2) hybridization of DNA from a single clone to arrays of oligonucleotides. A review of the thermodynamics and kinetics of oligonucleotide hybridization in solution reveals that these processes are the same, provided that the length of the cloned DNA in solution is reduced to the effective length of the cloned DNA on a solid support. Reducing the size of the strands would also decrease possible confounding effects of secondary structure. A variation on Format 2 involves release of hybridized cloned DNA fragments from oligonucleotides immobilized in a gel matrix. In this case, the reaction is governed by a pre-equilibrium (T,,,) and a rate-determining step involving diffusion through the gel. A. Mirzabekov indidated that one of the difficulties with SBH is duplicated short sequences in the cloned DNA. This difficulty may be reduced for longer DNAs either by using longer immobilized oligonucleotides or by using end-to-end continuous stacking of adjacent oligonucleotides hybridized for the DNA target. This increases the Td of the duplex formed and is equivalent to working with a much more complex matrix. For example, a matrix of immobilized octanucleotides used together with a preselected solution mixture of pentanucleotides can produce the same results as a matrix with immobilized tridecanucleotides. R. Crkvenjakov and R. Drmanac, using Format 1, and Mirzabekov, using Format 2 in a gel, demonstrated in dot blots and in arrays of cloned DNA the ability to distinguish hybridization of very short oligonuleotides to complementary sequences from hybridization to mismatched sequences or from nonspecific binding to background. J. Brown demonstrated the properties of synthetic nucleotides for inclusion in oligonucleotides that can substitute either for both purine (A and G) or for both pyrimidine (T and C) nucleotides. These molecules may prove useful for the practical synthesis of partially degenerate longer oligonucleotides for use in either format of SBH. (See discussion of gapped probes.) Oligonucleotide
Synthesis
K. Beattie described the multiple DNA synthesis technology that was developed in his laboratory at Baylor College of Medicine and subsequently commercialized by Genosys Biotechnologies, Inc. The segmented synthesis approach taken by Genosys employs stacks of porous Teflon wafers, each comprising a different reaction chamber containing derivatized support material, to simultaneously add a given base to all sequences within the stack. Following each reaction cycle (phosphoramidite method of synthesis), the wafers are sorted into different stacks to permit synthesis of a different se-
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1
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SPECIAL FEATURE quence within each wafer. Genosys’ current prototype instrument, automated in all aspects except for wafer sorting, is capable of synthesizing 100 oligonucleotides simultaneously with a cycle time of 6 min, including computer-directed manual sorting. The reagent cost at 0.2.hmol scale of synthesis is currently $0.25-$0.50 per base, and the product yield and quality are the same as for conventional automated DNA synthesizers. Automation of the Genosys machine is being continued with SBIR grant support from NIH. Beattie pointed out that if 10 of these Genosys machines were put to work synthesizing oligonucleotide libraries, the entire library of 65,536 octamers could be prepared in less than 2 weeks, and the entire library of 1,048,576 decamers could be synthesized in about 200 days. Efficient storage and retrieval of large oligonucleotides libraries are challenges that Genosys will address in a DOE-funded small business incentive research (SBIR) project. Beattie described some feasibilty studies that were performed in a collaboration between Baylor College of Medicine and Houston Advanced Research Center and aimed at fabrication of DNA chips containing hundreds to thousands of surface-bound probes acting as “genosensor” elements. Glass surfaces were derivatized by epoxysilane, then reacted with 5’aminoalkyl nonamer probes. The probes were shown to bind specifically at their Y-amino termini, spaced approximately 50 A apart on the surface, and when hybridized with 5’-32P-labeled 36-mer target DNA samples, gave good discrimination against internal mismatches. The HARC/BCM team, in collaboration with microfabrication engineers at MIT Lincoln Laboratory and a microrobotics collaborator, plans to produce miniaturized genosensors containing up to several thousand probes spaced 50-100 pm apart within a l-cm’ area. Beattie’s laboratory at HARC will be developing genosensor applications for genomic mapping and mutation detection. S. Fodor outlined the relative advantages and disadvantages of the off-chip and on-chip strategies for preparation of DNA probe arrays and described the approach being pursued by Affymax Research Corp., which utilizes addressable laser-activated photodeprotection in the chemical synthesis of oligonucleotides (or peptides) directly on a glass surface. Affymax scientists have recently developed new phosphoramidite derivatives capable of highly efficient light-activated detritylation and will now be evaluating these reagents for producing miniaturized DNA chips, including an octamer chip within a 1-in.2 area. E. Southern of Oxford University described his laboratory’s experience with SBH Format 2. They have produced DNA probe arrays by an on-chip strategy that employs a stable linkage comprising a 20.atom aliphatic chain attached to a glass surface with a primary hydroxyl group at the other end which is used to initiate synthesis of oligonucleotides in situ. Oligonucleotide arrays were built by directing the base additions to channels created by barriers between plates; alternate bases are added through channels placed at right angles to the previous addition, and finer spacing is used as the length of the oligonucleotides and the complexity of the set increase. Using this approach, a complete array of 4” s-mers is achieved in s cycles, e.g., 65,536 octamers in eight cycles. Thus far the Oxford group has made arrays of 4096 oligonucleotides on plates of 20 X 20 cm.
SBH Results:
Format
1
In the session on SBH Format 1 in which complex target DNAs are on a surface and the oligonucleotide is in solution, results were shown by five speakers from two groups: ICRF (headed by H. Lehrach) and Argonne National Lab (headed by Drmanac/Crkvenjakov). H. Lehrach presented a mapping strategy for linkup of cosmids via oligonucleotide fingerprint matching and announced imminent ordering of the S. pombe genome in cosmids. The reference library concept as an integral means of organizing information gathering in mapping and sequencing via shared filters with arrayed clones was presented, and the major role of oligonucleotide hybridization in this inquiry emphasized. New technology development in London is in the area of robotic formation of dense filter arrays from microtiter plate samples with more than the standard 96 wells. Pilot experiments with oligomers substituted with modified bases to enhance binding were presented by J. Hoheisel. A full commitment to a project for providing signatures for partial sequences of clones in cDNA libraries by SBH was announced. Crkvenjakov showed results of two model sequencing experiments, one starting with the known sequence and the other with the sequence not known to the investigators. In the blind test, the Argonne group determined in parallel the sequence of 111/116-bp stretches in three related inserts using a supplied probe set guaranteed to contain at least one subset that defined one of the sequences by the maximal overlap of its members. The results obtained with 164 probes uniquely defined the three sequences by positive hybridization of more than five probes per basepair on average. When the test results were evaluated, it was found that no wrong bases were called in 343 bp of hybridization-determined sequence. The maximal data collection error rate was 3.25%, and the real rate is probably lower. Drmanac presented results on the development of technological components of a hybridization data production line based on Ml3 clone libraries. Parallel clone placement, growth, sample preparation, and robotic spotting on filters in dense arrays based on microtiter plate format were shown to work at performance levels that allow collection of up to 10 million cloneprobe hybridization data per day. For example, 13,824 dots were made on an 8 X 12-cm filter by offset printing of samples from 144 microtiter plates. This extrapolates to a measurement capacity of hybridization signals from over 150,000 dots/ 30 min scanning of a single screen using a Phosphorimager instrument (Molecular Dynamics, Sunnyvale, CA). Lehrach and Crkvenjakov summarized the results on Format 1 SBH by concluding that the time is ripe for large volume data collection experiments both in mapping and in partial to complete sequencing. The potential for dissemination and immediate applicability of the technical developments in this area in a wide variety of genome-related problems was stressed by pointing out that SBH is the natural outgrowth of familiar and widely used methods of molecular biology. Probe and Sample SBH dominantly depends analysis. Six talks presented
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Design
on sophisticated in the informatics
computational section of the
SPECIAL FEATURE Moscow SBH meeting are a healthy sign of progress. The main problem of SBH is frequent occurrence of branching points, especially in data containing errors and in some sequences comprising short, partly degenerate tandemly repeated oligonucleotide sequences. The informatics solutions presented could reduce the required numbers of scored probe-clone hybridization data per basepair of genome sequence by up to 1000 times. One solution described by W. Bains is gapped probes (B,N,B, or B,I,,,B,, where B, and B, are unique base sequences, N,,, is a stretch of fully degenerate bases, and 1, is a stretch of m inosines). His results, and those of Yu. Lysov, P. Pevzner, and R. Lipshutz, demonstrate that gapped probes will have 10 times more power than ordinary probes. Lysov showed calculations revealing a high efficiency of solving branching points using the method of continuous stacking hybridization described earlier for a few additional hybridization cycles. In addition to modified probe design, higher efficiency can be obtained by mathematical solutions that operate with relative intensities of hybridization signals instead of assuming all or none hybridization. The efficiency of sequence reconstruction depends on a correct definition of the error function for the data. J. Elder demonstrated successful sequence reconstruction with low discrimination but highly redundant hybridization data. For sequencing complex genomes with extensive polymorphisms, SBH appears to be especially powerful. Drmanac presented data from a few small-scale simulation experiments, demonstrating efficient use of overlapped DNA fragments and comparison of similar sequences for solving branching points. As a consequence, 10 million to 1-4-kb-long genomic clones hybridized with 2000 6- to 8-mer probes may be sufficient for almost complete sequence reconstruction of the human genome. Drmanac’s view of the next step in SBH informatics is developing efficient software for unifying mathematical and heuristic solutions and optimizing the parameters in comprehensive simulation experiments on the megabase range. Drmanac concluded that appropriate combinations of conventional gel sequencing and SBH can be more efficient than any one method alone for genome sequencing. What is more important, genome sequencing with the two methods combined can be implemented immediately using SBH Format 1. SBH Results:
Format
2
Format 2 is based on hybridization of DNA to a matrix of immobilized overlapping oligonucleotides, identification of oligonucleotides that upon hybridization form perfect duplexes with DNA, and reconstitution of DNA sequence by searching for hybridized oligonucleotides that overlap with each other. U. Maskos described experiments with complete arrays (5 x 5 cm) of octapurine sequences. Hybridization with a complete set of 256 octapyrimidines was used to find conditions that minimize the effects of base composition on duplex yield. Tetramethylammonium chloride was found to remove much of the effect, but some sequence-dependent differences remain. The same data were used to calibrate the array in model sequencing and mutation analyses, using synthetic pyrimidine 20-mers. The algorithms developed by Elder, described below, produced the correct sequences. In a novel approach to the
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detection of sequence differences, two digital images produced by hybridizing two related sequences to the same array were subtracted. The sequence difference was derived from the difference data. Maskos also described small arrays (90 oligonucleotides) for optimizing the choice of allele-specific oligonucleotides (ASOs) used in diagnosis of genetic diseases. Using the welltried example of the sickle cell mutation in the P-globin gene, he showed excellent discrimination between wildtype and mutant alleles. Significantly, probes 110 nucleotides long, made from PCR products, behaved as well in hybridizations as synthetic 19.mers. An advantage of the glass plate technology is that ASOs can be laid down in stripes so that multiple probes can be laid across them in orthogonal stripes to introduce parallelism into the analysis. For complete sequencing, it could be necessary to have a matrix of all possible oligomers, for example, all 65,536 octanucleotides. Mirzabekov estimated that the method could be realized in 2-5 years depending on available resources and predicted that it will provide a fast and inexpensive sequencing procedure that could be easily automated and used in many laboratories for sequencing millions of bases per day. Partial sequencing or comparison of different sequences would need only partial matrices. This can be developed in 1-3 years. Diagnosis of genetic diseases and changes in the sequence of genes with known st.ructure would need a matrix of only tens to thousands of immobilized oligonucleotides complementary to the whole gene sequence or a part of it. This could be developed in a year, if based on PCR DNA amplification. Analysis could be performed rapidly and inexpensively. Mirzabekov demonstrated that discrimination of perfect duplexes from imperfect ones containing internal mismatches and often also terminal mismatches can be carried out by measuring hybridization intensities. The Moscow group has made progress in the manufacturing of a sequencing chip, a micromatrix of immobilized oligonucleotides. The use of gels fixed on a glass plate as a support for immobilization has been patented. Technologies have also been developed for making 30 X 30 to 100 X 100 ym gel squares that are lo-30 ,um thick, for applying oligonucleotide solutions to these. Chemistry for quantitative and fast immobilization has been developed. Modifications of these and other novel technologies are under development for commercial production of chips. At present, the Moscow group can produce chips containing tens of immobilized oligonucleotides. In the near future, chips with hundreds of oligonucleotides are expected. By using modified microelectronic technologies, it should be possible to produce thousands of chips containing hundreds of thousands of immobilized oligonucleotides at a cost of $1-50 each. The Moscow group is using fluorescent tags for DNA labeling and quantitative detection of hybridization. Fluorescence appears to be better t,han radioactive measurements in resolution, speed, safety, and other characteristics. Fluorescence can be used with the microchips described above. Prototype equipment for quantitative fluorescent measuring of hybridization has been constructed. It consists of a fluorescent microscope, a CCD camera, a thermostated chamber for hybridization, and a computer with relevant programs for image analysis and se-
SPECIAL FEATURE quence reconstruction operations. The cost of the fully automated equipment could be less than $50,000. Some major difficulties remain to be overcome. 1. Complexity ofthe chip: The longer the immobilized oligonucleotides, the higher the efficiency of sequencing, but the complexity of the chip needed grows exponentially. A chip with 65,536 immobilized octanucleotides would enable sequencing DNAs about 200-400 bases long. This matrix offers a compromise between efficiency and complexity. Mirzabekov suggested a procedure consisting of several rounds of hybridization of DNA with the same octanucleotide matrix in the presence of different short oligonucleotides (preselected by computer), such as pentanucleotides, to reach the efficiencies typical of a matrix with immobilized 13.mers. This will enable efficient sequencing of DNAs 3000-5000 nucleotides long, in a few minutes. The method is based on continuous stacking hybridization illustrated earlier. 2. Different stability of A-T and G-C basepairs in DNA duplexes: Generally, hybridization measurements need to be carried out at different temperatures since duplexes with different A-T basepair contents have different thermostabilities. By using gel-immobilized oligonucleotides, the Moscow group found that the thermal stability of duplexes depends on the concentration of immobilized oligonucleotides. However, at a properly chosen concentration of immobilized oligonucleot.ides. it becomes possible to build up a normalized matrix where all oligonucleotides form duplexes with similar or even equal thermostability despite differences in their A-T contents. 3. DNA size and its secondary structure: Long DNA fragments can form secondary structures that interfere with hybridization. Therefore, fragmentation of DNA to rather short fragments is necessary, preferentially with a narrow size distribution. Several approaches for DNA fragmentation to specific size have been developed in MOSCOW. Informatics The highlights of the informatics section of the SBH conference in Moscow were new ideas for modeling hybridization, fragment reconstruction, and sequencing chip design. J. Elder from Southern’s group at Oxford presented a linear model for going from sequence to detected SBH signal. The model is given by D = R(F) + s*e, where D is the observed data. R is a linear response function, F is the multiplicity of each oligonucleotide in a candidate fragment, s is the standard deviation of the noise, and e is a multidimensional normal variable. If D, F. and e are vectors, then R is a matrix and s is a scalar. Elder went on to describe an implementation of the model that derives R and s from the data using maximum likelihood and maximum entropy considerations. An example using data from Southern’s chip was shown. This general model and the associated parameter estimation techniques provide a solid basis for further development of the hybridization models. R. Hagstrom from Argonne National Laboratory introduced a new methodology for fragment reconstruction. Specifically, this new methodology takes the hybridization data and yields a directed Eulerian multigraph. (This is a series of points [vertices] connected by multiple paths with directions assigned to
each path, such that all the paths can be traced without any duplication. An example is a solution to the classic mathematical problem: the seven bridges of Konigsberg.) Possible fragments correspond to Eulerian paths in the graph. Suppose G is an Eulerian multigraph, and D is the hybridization data; then let UD 1G) be the likelihood that any fragment G corresponding to G yields data D. Then, given hybridization D, the new algorithm finds a graph G that maximizes L(D / C). A specific implementation of the algorithm for linear response function and normal errors was presented. It was shown that this algorithm has polynomial complexity. This new algorithm significantly advances work on the problem of fragment reconstruction. It subsumes the previous work of Pevzner and Lipshutz and is flexible enough to allow significant modification of the definition of L(D 1G). This will allow adjustment of the method as our understanding of the hybridization process improves. Bains, Lipshutz, and Lysov presented work on the selection and design of a highly efficient sequencing chip. The concept of a probe was extended to be a set of oligonucleotides colocated on a chip or applied to all targets simultaneously. A chip is then a collection of such sets. For a fixed number of probes, the efficiency of a chip can be measured by the maximum length of a fragment such that the probability of branching on reconstruction is less than a fixed probability. Bains described simple probes with internal gaps. Lysov described families of such probes. Lipshutz described three very general sets of probes called gapped (described earlier), alternating (for example, ANGNGNC where N is any base), and binary (for example, YRRYYRY, where Y is pyrimidine and R is purine). Efhciencies for the binary family were described. It was shown that for a fixed capacity the efficiency varies with the square root of the probability of branching for binary chips while it varies linearly in the case of standard probes. These results point the way to being able to unambiguously sequence longer fragments using the same or a smaller number of probes. Bains also presented an initial analysis of the probability of repeated 8- tuples as a function of their separation. This analysis was performed using the human hemoglobin locus. The function is damped and periodic. This is a surprising result and is quite different from the result using random sequence data. This result will have significant impact on predicting the efficiency of SBH. In particular, future SBH simulations must be performed using actual sequence data. Lipshutz present updated work on maximum likelihood fragment reconstruction. Simulated results based upon the improved model were shown. Drmanac presented his earlier work on heuristics for fragment reconstruction.
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Hybridization
Detection
Detection techniques presented ranged from low cost/moderate sensitivity to high cost/high sensitivity. Specifically, four detection schemes were presented. The first technique was conventional radioactive labeling, which is a proven technology yet not amenable to miniaturization due to the l-mm resolution limit. Next, the optical approach that relies on fluerescent tags serving as markers for hybridized DNA was presented. Early results indicate at least a 3:l discrimination factor for hybridization detection. Such approach is amenable to
SPECIAL FEATURE miniaturization, yet requires costly laser optical equipment. The third approach presented was derived from mass spectroscopy. This approach entails vaporizing the surface of a stable isotope-labeled sample with a probe beam scanned in a 2-dimensional pattern and subsequently detecting the composition of the sample with a tuned laser. The discrimination factor for such an approach exceeds lOO:l, with an incredible sensitivity of a few parts in a trillion, basically detecting a few atoms per isotope. The trade-off for such sensitivity was seen to be the cost in supplying the powerful laser equipment. The last technique described is a direct detection approach, in which no tags are required to mark the samples for improved discrimination. A microelectronic detection technique
that relies on a change in the dielectric properties in a localized DNA probe test well was introduced, constructed using standard microfabrication techniques. The microelectronic approach provided a 1O:l discrimination factor in preliminary hybridization experiments. Moreover, the method has the potential of providing an extremely low-cost solution to hybridization detection, since the accompanying instrumentation can be placed directly on the microchip with the hybridization array. Overall, the potentially viable hybridization detection techniques discussed were summarized in terms of maturity, speed, cost, resolution, preparation, training, hazard, and instrumentation.
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