Patent Application: US-14048002-A

Abstract:
a method for assembling nucleic acid sequence fragments is disclosed . the fragments are assembled using information about their relative position inferred by comparison of the fragments against a known sequence of a related nucleic acid . additionally , the method localizes fragments to bacterial artificial chromosomes and determines relative position of bacterial artificial chromosomes using sequence comparison information . the method utilizes the information about relative orientation , mutual distance , fragment localization to bacterial artificial chromosomes , and relative position of bacterial artificial chromosomes to constrain the assembly process , thus resulting in a more accurate assembly requiring fewer sequencing reactions .

Description:
the term “ genome deciphering ” has often been used to summarize newly completed dna sequencing projects . however , the true deciphering will require the development of an understanding of the function of dna sequence , an effort that will take decades . to decipher biological function of dna sequence , a number of approaches are currently pursued . the most powerful of them involves yet more dna comparative sequencing of related species ( ref . 25 ): “ probably the most powerful tool to identify the coding exons , as well as the regulatory regions , is a comparison of the sequence across different genomes for that purpose , full - scale sequencing of the laboratory mouse genome already has been initiated , and the sequencing of the rat and zebrafish genomes will not be far behind . in both the public and private sectors , serious consideration is being given to the sequencing of other large vertebrate genomes , including the pig , dog , cow , and chimpanzee . . . . ” the more similar the reference nucleic acid sequence to the sequence of the original nucleic acid , the more effective will be the method disclosed in the present invention compared to the classical olc process . as the number of sequenced genomes increases , the utility of the disclosed method will rapidly increase . in particular , because the similarity of genomic sequences of primates exceeds 90 %, the assembly of the genomes of chimpanzees and other primates will be greatly improved through the use of the human reference sequence . even genomes of not so closely related species such as humans and mice share an average 85 % similarity in biologically important protein coding exon regions ( ref 20 ), thus opening the opportunity to save considerable effort in the sequencing of these important regions in mice by applying the disclosed method . another obvious immediate utility of the method is in the sequencing of genomes of different organisms within the same species where the reference genome sequence for the species is already known . genomic sequence similarity across different organisms within a species , such as humans , typically exceeds 99 %. the classical olc method for sequence assembly ( ref 16 , 20 , 21 , 24 ) utilizes the following pieces of information in the assembly process : ( 1 ) sequences of fragments of nucleic acids ; ( 2 ) sequences of known repetitive elements ; ( 3 ) information about false fragment overlaps ; ( 4 ) information about the approximate distance between pairs of fragments ; ( 5 ) information about localization of fragments within the same subregion ( bac ); and ( 6 ) information about the relative position and overlaps between subregions ( bacs ). it is important to note that the source of all the information utilized by the classical olc method and listed above is the original nucleic acid sequence . in contrast , the disclosed method augments or completely replaces the need for this information by employing the information obtained by comparisons of fragment sequences against another known reference sequence or a scaffold . the present method assumes that a number of fragments are obtained from the first nucleic acid . second ( reference ) nucleic acid , or a set of other nucleic acids are then identified that satisfy the following two criteria : ( 1 ) sequence fragments , complete sequence or sequence scaffolds from the second nucleic acid are available and ( 2 ) it can be reasonably assumed that there is similarity between the sequences of the first and second nucleic acids . prior to inferring positional information , calibration of the method needs to be performed by setting the threshold t . the threshold is used to estimate whether or not a fragment from the first nucleic acid has an orthologous match in the second nucleic acid : if the match exceeds t , the match is declared to be orthologous , if the match falls below t , it is declared non - orthologous . in order to set the threshold , the degree of similarity between the first and second nucleic acid is first estimated . the degree of similarity is measured by aligning known orthologous sequences from the two nucleic acids . if the exact orthologs are not known , long stretches of genomic dna may be used as surrogates . the comparisons should yield the information about the expected percent similarity between orthologous regions . the information about the percent similarity between orthologous regions is then used to set threshold t of percent similarity so that most ( preferrably 85 %) orthologous matches fall above the threshold . it should be noted that the threshold t may be based either on the comparison of nucleic acid sequences or on the comparison of inferred amino acid sequences , where appropriate . typically , one threshold would be appropriate for amino acid coding regions while another threshold would be appropriate for other regions . once the threshold is initially determined , it is further adjusted for accuracy of positional information . for that purpose , a set of mate pairs needs to be used . the mate pairs are compared against the second nucleic acid in order to identify matching mate pairs . a mate pair identified as matching the second nucleic acid sequence if both of the mates match the second nucleic acid above threshold t . next , the distances between the matches in the second nucleic acid (“ inferred distances ”) are compared against the expected mate pair distances (“ measured distances ”). since the measured distances are known only to a certain degree of accuracy ( ref 16 ), a mate pair distance is considered to be correctly inferred if it falls within three standard deviations from the measured distance . threshold t is then further adjusted so that most ( preferrably 80 %) of distances are correctly inferred . in addition to calibrating the threshold t , the comparison of inferred and measured distances is used to estimate the confidence interval for the inferred distance . the confidence intervals are essential for use of inferred distance information by the greedy path - merging method ( ref . 16 ). if a sequence fragment matches the reference sequence above the threshold t , a match is declared and the inferred distance is reported . in the following , the inventor considers methods for inferring particular types of positional information about fragments based on matches of fragments against the reference sequence . the first type of information that is inferred is the information that a fragment is localized within a subregion . in the prior art , this information is obtained exclusively through the clone - by - clone shotgun method by tracking the clone of origin for each fragment . a method for inferring localization of fragments by comparison to second nucleic acid is illustrated in fig1 . the first step in the process is to identify a set of fragments that are known to fall within a subregion of the first nucleic acid . for example , such set of fragments may be the fragments obtained from the same bac clone of insert size 200 kb . in the second step the fragments are compared against the reference nucleic acid . in the third step a position (“ subregion center ”) of the reference nucleic acid is calculated by averaging positions where fragments match reference nucleic acid . center of subregion ( 101 ) is a position in the reference nucleic acid . radius ( 102 ) is then defined as half the estimated size of the subregion from the original nucleic acid ( in case of a bac from our example , the radius is 100 kb ). finally , if a previously unlocalized fragment ( 103 ) from the first nucleic acid matches reference sequence within the radius of the center of subregion , it is inferred that it also falls within the subregion of the original nucleic acid ( in our example , it would be inferred that the fragment also falls within the bac ). the second type of information that is inferred is the information about the overlap of two fragments . in the prior art this information cannot be obtained with certainty based on sequence similarity of fragments due to the potential presence of repetitive elements and repeat - induced similarities between fragments . a method for inferring overlap of fragments by comparison to second nucleic acid is illustrated in fig2 . in the first step , overlap of two fragments ( 201 and 202 ) from the original nucleic acid is inferred if they match in an overlapping fashion a region ( 203 ) in the reference nucleic acid ( 204 ). such overlap is considered “ confirmed ”. in the second step , two fragments whose overlap is confirmed may be replaced by a single fragment that results from the assembly of the two fragments . the third type of information that is inferred is the information about the distance between two fragments . in the prior art this information is obtained through the sequencing of mate pairs and involves significant additional experimental and logistic effort . a method for inferring fragment distances by comparison to second nucleic acid is illustrated in fig3 . distance between two fragments ( 301 and 302 ) from the original nucleic acid is inferred as the distance ( 303 ) between unique matches ( 304 and 305 ) of the two fragments in the reference sequence . if a match of a fragment is not unique , the strongest match is considered , but only in case the strongest match is significantly better ( say , at least 5 % better ) that the second - strongest match for the same fragment . if any of the fragments does not have a unique match , or an acceptable strongest match , distance between fragments is not inferred . the inferred distance is accompanied by a confidence interval ( plus or minus three standard deviations from the estimated distance ). the standard deviations are estimated empirically based on a control set of fragments of known distance in the first nucleic acid . the deviation is calculated based on the observed differences of the inferred distance from the true known distance under an assumption that the differences are normally distributed . the fourth type of information that is inferred is the information about false fragment overlaps . such overlaps are typically induced by the presence of a previously unknown repetitive element that occurs at both the end of one fragment and at the beginning of another fragment . false overlaps cause significant assembly problems and significant effort has been directed toward at least partially alleviating the problem . present invention utilizes the information present in the reference nucleic acid in order to alleviate the problem of false overlaps . a method for detecting false overlaps is illustrated in fig4 . first , the fragments are tested for the presence of known repetitive elements . if the presence of a known repeat is established and if the similarity between fragments can be explained by the presence of a repetitive element , false overlap is considered detected ; otherwise , move to the next step . in the next step , the matches of the two fragments against the reference sequence are considered . if the two fragments both match the reference sequence at a distance ( 404 ) that precludes overlap , false overlap is detected ; otherwise , no action is taken . the fifth type of inferred information are the sequences of previously unknown repetitive elements . this information is obtained as a by - product of the method for detecting false overlaps , as is illustrated in fig4 . in the first step , presence of a repetitive element is inferred by detecting the situation where the end sequence ( 403 ) of one fragment matches the beginning sequence ( 402 ) of the other fragment but the fragments are inferred to occur at a distance by comparison against the reference nucleic acid . in the second step , check whether the match of the fragments to the reference sequence is complete or only partial ( with non - matching overhangs ). if the match is partial , the non - matching parts of the fragments comprise the sequence of a repetitive element . if the match is complete , then the region of similarity between the two fragments is detected as the sequence of a repetitive element . the sixth type of information that is inferred is the information about the overlap of two subregions from the first nucleic acid ( say bacs ). in the prior art this information requires costly fingerprinting experiments and cannot be obtained with certainty ( ref 20 , 21 ). a method for inferring overlap of subregions by comparison to second nucleic acid is illustrated in fig6 . in the first step , overlap of two subregions ( 601 and 602 ) from the original nucleic acid is inferred if sequence fragments derived from both subregions match in an interleaved or overlapping fashion a region ( 603 ) in the reference nucleic acid ( 604 ). the minimum requirement for interleaving is that a match of a fragment from first subregion occurs between matches of two fragments from the second subregion or vice versa , or , alternatively , that two fragments , each representing a separate subregion , match the reference nucleic acid in an overlapping fashion . in the second step , two subregions whose overlap is inferred at high confidence are replaced by a single subregion that results from the assembly of the two subregions . one should note that fragments are typically matched in both the direct orientation and as reverse complements . fragments may be matched either at the nucleic acid sequence level or as translated amino acid sequence . the amino acid sequence may be translated in three different frames and in direct and reverse - complement orientations of the nucleic acid , thus resulting in six translated amino acid sequences per single nucleotide sequence fragment . a variety of well - known sequence matching and alignment methods ( as described in ( ref 10 )) may be used for the purpose of finding matches between sequences of fragments and the sequence of the second nucleic acid , including blast , fasta , and dynamic - programming based methods such as global alignment and smith - waterman alignment . in particular , the widely used fasta method ( ref . 26 ) is useful , especially for amino acid sequence comparisons , because of its speed and its sensitive alignment of longer fragments . while a reference nucleic acid sequence , such as a complete genomic sequence , is an ideal reference for the disclosed method , scaffolds are used instead in situations where they are available and where the complete reference sequence is not available . scaffold comparison is performed by the method described in ( ref . 18 ). while the different types of positional information outlined above can be obtained from an individual reference sequence , multiple reference sequences or scaffolds , or combinations thereof are used whenever available . if multiple references are used , distances inferred from individual references are combined pooled by calculating a mean value . standard deviations and confidence intervals are combined by applying well - known statistical techniques for combining multiple measurements of the same quantity in the presence of measurement error . the inventor here describes how is the positional information used in the context of a large genome sequencing project . the block diagram in fig5 describes the information flow in the disclosed method . the portion of the diagram to the left of the dotted line in fig5 illustrates the methods of inferring positional information while the portion of the diagram to the right illustrates the hierarchical olc sequence assembly process . it should be recognized that smaller - scale sequencing projects consist only of a subset of steps outlined in the diagram in fig5 . for example , bac - level sequencing would not require the global assembly step ( 507 ). nevertheless , the inventor here describes the most difficult process in order to employ all the kinds of inferred positional information that described in sections b , c , d , e , f , and g . the portion of the diagram in fig5 to the left of the dotted line illustrates the methods of inferring positional information based on reference sequence ( 501 ), the six grayed blocks correspond to the six methods for inferring positional information disclosed in sections b ( block 510 ), c ( 540 ), d ( 520 ), e ( 550 ), f ( 530 ), and g ( 560 ). localization information ( 511 ) is inferred ( 510 ) as described in section b and the newly localized fragments are simply added to the appropriate bacs ( 512 ) for processing by the screener / overlapper ( 504 ) and the bac - level assembler ( 505 ). distance information ( 521 ) is inferred ( 520 ) as described in section d and the newly inferred fragment distances are simply added to the appropriate mate pair distance information ( 522 ) for processing by the screener / overlapper ( 504 ) and the bac - level assembler ( 505 ). the information about new repeats ( 531 ) is inferred ( 530 ) as described in section f and the newly inferred repeat sequences are simply added to the database of known repeat sequences ( 533 ) for processing by the screener / overlapper ( 504 ). the information about confirmed overlaps ( 541 ) is inferred ( 540 ) as described in section c and made available for use by the screener / overlapper ( 504 ), and the confirmed overlaps that come from a particular bac are used to assemble the shorter fragments into a longer fragment and thus increase the phase ( from 0 to 1 ) of individual bacs ( 512 ). the information about repeat - induced overlaps ( 551 ) is inferred ( 550 ) as described in section e and made available for use by the screener / overlapper ( 504 ) and the bac - level assembler ( 505 ). the information about subregion ( bac ) overlaps ( 561 ) is inferred ( 560 ) as described in section g , is simply combined with the overlap information obtained by physical mapping ( 562 ), and then made available for use by the genome - level assembler ( 507 ). the portion of the diagram in fig5 to the right of the dotted line illustrates a hierarchical olc sequence assembly process that utilizes previously described components used in the assembly of genomes of drosophila ( ref . 8 ) and human ( ref 20 , 21 ). bac shotgun sequencing ( 502 ) and whole - genome shotgun sequencing are performed using standard methods ( ref . 20 , 21 ). the screener / overlapper ( 504 ) performs the two - step function of first screening sequence fragments for the presence of repetitive elements and , second , detecting overlaps between fragments for use in the subsequent layout stage . if the available hardware and processor speed are the bottleneck in a genome scale project , an appropriate screener / overlapper for genome - scale projects has been described in ( ref 8 ), otherwise , the use of blast searches for both repeat screening in the screener step and overlap searches in the overlapper steps is appropriate . an appropriate setting for the screener ( ref . 8 ) is “ hard screen ” and an appropriate threshold for detecting overlaps by the overlapper is fewer than 6 % differences in 40 bp unmasked ( unscreened ) sequence , as described in ( ref . 8 ). the inferred information ( 541 , 551 ) is combined with the information produced by the screener / overlapper using the following rule : each inferred overlap ( 541 ) is simply added to the set of overlaps detected by the overlapper . each detected false overlap ( 551 ) is looked up in the list of overlaps produced by the overlapper and , if found , is deleted from the list . an appropriate method for the bac - level assembler ( 505 ) step is the greedy pathmerging method , as disclosed in ( ref 16 ). the greedy path - merging method smoothly integrates the shotgun fragment information at the bac level with the genome - level shotgun information , including mate pair distances . one of the features of the greedy path - merging method is that it breaks spurious assemblies at the bac level (“ bactigs ”). the only straightforward modification required of the greedy path - merging method is that it should break the bactigs that contain false repeat - induced overlaps detected by comparisons against the reference sequence ( 551 ). otherwise , the method should be applied as described in ( ref . 16 ). the breaking of erroneous assemblies due to repeat - induced overlaps ( 551 ) should significantly improve the overall quality of assemblies at the bac level . global assembler method merges bac - level assemblies into a global assembly guided by a physical bac map ( 562 ). note that the inferred bac overlap information ( 561 ) is simply added to the physical map information ( 562 ) prior to the processing by the global assembler . thus , no changes to the genome - level assembler are necessary in order to accommodate the inferred positional information . depending on the degree of fragment coverage and other factors , the output ( 508 ) of the genome - level assembler consists either of a complete sequence or of a number of sequence scaffolds , each scaffold consisting of a number of mutually oriented sequence contigs . an appropriate embodiment of the genome - level assembler ( 507 ) is the four - step method outlined in fig6 of ( ref 20 ). the gigassembler method ( ref 24 ) performs this set of four steps but it also uses additional information such as messenger rnas and ests . in case such additional information is available , gigassembler is an appropriate embodiment . note that in cases where the second ( reference ) nucleic acid and the first ( to be assembled ) nucleic acid share significant similarity , such as , for example , the similarity between the genomic dna of humans and chimpanzees or between the genomic dna of different organisms within the same species , the inferred positional information may completely substitute the information ordinarily obtained by other means . in other words , a method illustrated in the diagram in fig5 may in this case be modified by eliminating blocks ( 502 ), ( 533 ) and ( 562 ) and by replacing “ bac ” by “ subregion ” and by omitting the phrase “ and mate pair distances ” from block ( 522 ) and the method would still work . in other words , with the help of the reference sequence a true “ genome level shotgun ” method where neither clone - by - clone sequencing nor mate pair information is tracked would be feasible . finally , while the diagram in fig5 illustrates a complete genome - level sequencing method , parts of the diagram may also be performed independently . for example , in order to achieve bac - level assembly one may omit the genome - level assembly step ( 507 ). indeed , if the fragment coverage is very high ( say 15 - fold ) and if the great majority of bacs fed into ( 507 ) are phase 3 ( filly assembled sequence ), the whole - genome level assembly problem becomes quite trivial . it should be noted that the choice of assembly programs to be used in conjunction with the present invention depends on the circumstances of the particular application . discussions of the advantages and disadvantages of particular methods abound in the cited literature ( ref . 1 , 5 , 8 , 13 , 16 , 19 , 21 , 20 , 24 ). published information needs to be taken into account when deciding which assembly method or a combination of methods is most appropriate for the particular application . the following is a practical guide for selecting the most appropriate method : if the sequence to be assembled does not exceed 3 mb and redundancy of coverage exceeds 7 ×, then apply euler method ( ref . 19 ), otherwise , if the coverage is less than 7 ×, apply cap3 ( ref . 5 ). if the project is whole - genome sequencing of genomes significantly larger than 3 mb , and if bac - level shotgun is combined with whole genome shotgun including mate - pair information , apply greedy path - merging method to improve bac - level assembly ( ref 16 ) and then apply the gigassembler method ( ref . 20 , 24 ) to combine bac - level assemblies into a genome - scale assembly . otherwise , if a whole - genome eshotgun approach is pursued and mate - pair information is available , apply the whole genome assembly method ( ref . 8 , 21 ). in as much as the information inferred by the methods described in the present invention is identical in form to the information otherwise utilized by the assembly programs , the information inferred by the present invention is simply added to the other existing information to improve the assembly process . in one embodiment the inferred distance information is identical in form to the mate - pair information and is directly utilized by the greedy path merging method ( ref 16 ). in yet another embodiment , the inferred bac localization is identical in form to the bac localization inferred by the bac - level shotgun method and is used directly in bac - level assembly by cap3 ( ref 5 ) or the greedy path - merging method ( ref 16 ). in a further embodiment , the inferred information about fragment overlaps is used to replace two short fragments by a longer fragment obtained by merging the two short fragments and thus improve the performance of the euler assembler by resolving “ tangles ” ( ref 19 ). in a yet further embodiment , the inferred information about repeat - induced false overlaps is directly used in the greedy path - merging method for breaking up erroneous bactigs ( ref 16 ). in an even further embodiment , the inferred information about newly detected repeated sequence is used by the the screener / overlapper ( ref . 8 ) in order to detect and tag repeat - induced overlaps prior to the layout stage of the assembly process . in a still further embodiment , the inferred information about bac overlaps is added to the bac overlap information obtained through restriction fingerprint analysis to improve the assembly produced by the gigassembler method ( ref 20 , 24 ). it should be noted that different assembly programs utilize different subsets of the positional information that is inferred as described in the present invention . for example , euler assembler does not utilize the mate pair or distance information ( ref . 19 ). a practitioner with ordinary skill in the art should be able to competently decide which types of inferred information , as described in the present invention , are usable by the particular assembly program . p the accuracy of inferred information may vary . the inferred information is useful despite the fact that it is not perfect . the assembly programs currently in use tolerate certain level of error in the input and implement methods that in effect assemble sequence using preponderance of evidence . it should be noted that in cases where the second ( reference ) nucleic acid and the first ( to be assembled ) nucleic acid share significant similarity , such as , for example , the similarity between the genomic dna of humans and chimpanzees or between the genomic dna of different organisms within the same species , the inferred positional information eliminates the need for information ordinarily obtained by other means . in one embodiment the inferred distance information eliminates the need for the mate - pair information and is directly utilized instead of the mate - pair information by the greedy path merging method ( ref 16 ), thus enabling whole - genome shotgun assembly without mate - pair information tracking . in yet another embodiment , the inferred subregional localization of fragments to subregions of size 10 kb to 10 mb eliminates the the bac localization inferred by the bac - level shotgun method and is used directly in subregion - level assembly by cap3 ( ref 5 ) or the greedy path - merging method ( ref 16 ), thus enabling whole - genome shotgun method without the need for bac - level shotgun sequencing . in a still further embodiment , the inferred information about bac overlaps eliminates the need for other bac overlap information , thus enabling tiling - path construction and global assembly by the gigassembler method without the step of restriction fingerprint analysis of individual bacs ( ref . 20 , 24 ). from the point of view of sequence assembly , the effort saved through application of the disclosed method may be measured by the decrease in the number of fragment sequencing experiments that need to be performed for assembly of the sequence of the first nucleic acid . by applying the lander - waterman model ( mathematical model establishing relationships between key variables in genome mapping ) one can calculate that in the case of the mapping of genome of bacterium e . coli 20 % of effort would be saved while in the case of the mapping of genome of yeast c . cerevisiae roughly 60 % effort can be saved . lander - waterman analysis does not apply to many higher eukaryotic genomes ( such as human and mouse ) due to presence of a large number of repetitive elements . repetitive elements impede sequence assembly by causing sequence similarity between non - overlapping fragments . such similarity may lead to assembly errors or to ambiguous ( incomplete ) assemblies . the method disclosed herein is uniquely suited to overcome the problem with repetitive elements due to the fact that it relies on inferred information about relative position of sequenced fragments , thus in effect creating “ bridges ” across the repeat - rich regions that are hard to assemble . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference : ( ref . 1 ) j . k . bonfield , k . f . smith , and r . staden . a new dna sequence assembly program . nucleic acids research , 23 : 4992 - 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