JP2001505780A - Quick gap closure method - Google Patents

Abstract

  (57) [Summary] Methods for gap closing and flanking sequence assembly of organisms using high density grids from a series of random genomic libraries prepared from isolated DNA of selected organisms are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION                           Quick gap closure method Related applications   This application is related to US Provisional Application No. 60 / 032,555, filed December 12, 1996. Claim your rights. Field of the invention   The present invention provides for the use of high density arrays or grids of genomic libraries A simple, cost-effective method for closing gaps that occur during genome random sequencing. It is a method that is effective in terms of cost. The method uses a full-length genome obtained from a partial gene sequence. It is also useful for rapid isolation of sequence sequences. Therefore, such a genomic sequence is Including the coding region. The method of the invention can be used to validate computer-generated assemblies. Is also useful. That is, it is useful for confirming the order or assembly of adjacent sequences. You. The method provides an alternative to chromosome walking. Background of the Invention   Advances in DNA sequencing technology and computer methodology are The speed at which a sequencing project could proceed was drastically changed. In particular, Flei schmann et al. (Science, 1995, 269: 496-512) describe whole-genome random sequencing and It shows that complete and complete organism assembly can be done in just a few months. this The random “shotgun” approach involves inserting a sequenced library. Input size, organism genome size, number of clones sequenced and salt sequenced Predict some sequence gaps between assembled sequences based on total number of groups I do. These gaps represent genetic gaps not identified by random sequencing. Corresponding to the intra- or intergenic region (eg, E.S.Lander and M.S.Waterman, " Genomicmapping by fingerprinting random clones: a mathematical analysis ”, Genomics, (1988), 2: 231-239). Alignment of adjacent sequences and completion of gap closure Typically, primers intended for all combinations of physical gap ends Performed by mer-based genomic PCR. However, this method is very Time consuming, labor intensive, 5-10 times more than random sequencing It can take a long time.   Therefore, adjacent sequences are aligned in random sequencing of the whole genome of an organism. There is a need for a more efficient way to complete gap closure. Summary of the Invention   In one aspect, the invention uses a high density grid of a genomic library, High-throughput sequencing and gaps in genome sequencing projects Methods for closed and flanking sequence assembly or clone alignment are provided. The method constructs a series of random genomic libraries for the selected organism And preparing a grid for each library, Lid immobilizes multiple clones from the library in a given area on its surface Have one surface. With this gap closure, the first random sequencing The complete sequence of the partial gene or gene not found in You. After assembling the flanking sequences of the partial gene sequence, the non-overlapping A hybridization probe corresponding to the end is prepared. Then, remove the probe Nucleotide hybridized to gridded library and assembled The nucleotide sequence spanning the non-overlapping ends of the nucleotide sequence is identified.   Other objects, features, advantages and aspects of the present invention will be apparent to those skilled in the art from the following description. It will be. However, the following description and specific examples will be helpful in understanding the present invention. Although a specific example is shown, it should be understood that the example is merely given as an example. It is. Within the spirit and scope of the disclosed invention, various changes and modifications may be made From reading the description of, and from reading other parts of the disclosure, It will be easily understood. Detailed description of the invention   Gene identification, sequencing and analysis are major goals of current scientific research . Identifying genetic sequences, determining their sequences, and their biochemical functions By applying recombinant technology, valuable gene products, such as , Proteins and peptides can be produced in large quantities. In addition, genetic Knowledge of the offspring sequence can be attributed to inappropriate expression and / or repression of the selected gene. Or the effects of external factors, such as carcinogens or teratogens, on gene function Diagnosis, prediction and cure of various disease states in plants and animals characterized by Can provide the key to treatment.   Currently, a method for whole random sequencing and assembly of complete organisms Exists. However, genomic sequence gap closure and flanking sequence alignments are not complete. The method required to complete is time consuming and labor intensive.   The present invention provides a high throughput gap closure useful in whole genome random sequencing. Methods are provided for strand and flanking sequence assembly. The present invention provides Sequence reactions, using multiple high-density grids prepared from genomic libraries Perform gap closure and flanking sequence assembly. The method of the present invention It provides a faster and more cost-effective means for sequencing nom. The method also provides for the full-length gene of a gene in which only a partial sequence is obtained by random sequencing. It also provides a quick means to obtain nome sequences. I. Definition   Some words and phrases used in this specification are defined as follows. RU:   As used herein, the term “gene” refers to the genome from which the cDNA sequence is derived. Nucleotide sequence. The term gene is, classically, processing Sometimes it refers to a genomic sequence that can produce different cDNAs, for example, by splicing. To taste. However, for the sake of simplicity, some of the full-length Is also referred to simply as a gene in this specification.   "Isolated" is defined as being changed "by the hand of man" from its natural state. That is, in the case of a natural product, it is changed or removed from its natural environment or both. Meaning that For example, a polynucleotide naturally occurring in a living organism or Is a substance that is not "isolated" but coexists in its natural state As used herein, the same polynucleotide or polypeptide separated from The term is "isolated". For example, for a polynucleotide, The term isolated means isolated from natural chromosomes and cells I do.   The term “organism” includes, but is not limited to, bacteria (gram negative and Virus, lower eukaryotic cells (e.g., fungi, yeast and filamentous fungi) , Single multicellular organisms (such as slime molds) and complex multicellular organisms including humans It is meant to include living organisms.   As used herein, the term “solid support” is derived from a genomic library. Immobilized by any available method, the solidified poly Hybridization of nucleotide sequence with other polynucleotides in sample Means any known substrate that is useful for making Many available One of the preferred solid supports is the International Patent Publication published May 30, 1991. A support described in Patent Application No. WO91 / 07087. Other useful supports Examples of, but not limited to, nitrocellulose, nylon, glass, silicone Rica and Pall BIODYNEC are included. Further modifications to conventional solid supports Good may be applied in the present invention.   The term "grid" refers to the anchoring of limited material in a genomic library It means a two-dimensional structure on the solid support to be immobilized.   As used herein, the term “predetermined region” refers to one or more specific clones. Or multiple copies are immobilized thereon and correspond to the sample nucleotides of the clone. When hybridization occurs, the clone Means a localized area on the surface of the solid support that allows hybridization You.   "Immobilized" refers to the anchoring of a gene to a solid support. The means of immobilization is our business Are known and customary to the individual and depend on the type of support used. II. Organism of the present invention   The present invention relates to a full-length genomic sequence and adjacent sequence assembly in genome sequencing. Genome ligature densification as a means of high-throughput gap closure, including brie Based on array usage.   A. Preparation of genomic library   For this analysis, a series of random genomic libraries for selected organisms Is prepared. Each library is a fraction of the size of the selected set of inserts. And ligated genomic DNA. To build these libraries, Sa mbrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .; Cold Spring  Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 Genomic DNA from the selected organism is obtained using standard methods in such molecular biology. First isolate. Then, Fleischmann et al. Science, 1995, 269: 496-512 The isolated DNA is randomly sheared according to the method described in (For example, by sonication, partial restriction enzyme digestion, partial DNAase digestion, etc.), modification And ligated into a plasmid or phage vector.   For example, in one embodiment, the average size of the insert is 1.0 and 5.0. The genomic DNA is fractionated to be between kb and A small insert library by ligation into a vector . A plasmid vector useful for constructing this small insert library Examples of, but not limited to, pBLuescript, Lambda ZAPII (Stratagene, LaJolla, CA), pUC19 and M13mp18 / 19 (New England BIOLABS, Beverly, MA) Included.   In this embodiment, the insert size is between 10 and 100 kb on average. Large insert libraries are also constructed with cosmid vectors. You. Cosmid vectors useful for constructing this large insert library Examples of, but not limited to, pLorist, pWEIS (Statagene, La Jolla, CA ), Lambda DASH2 (Statagene) and lambda GEM-12 (Promega, Madison, WI) are included. In addition, a medium series of inserts averaging about 5.0-10 kb in size. Insert libraries can also be used to fractionate genomic DNA into cosmid vectors. And can be prepared by   The final number of transformants for each library is Lander-Water According to the man theory (E.S.Lander and M.S.Waterman, Genomics, (1988), 2: 231-239) The final ligation product is transformed into E. coli (4-6 times the estimated number of coverages). Electroporate bacteria such as E. coli. Transformants Placed in microtiter plate and recognized as normal for certain bacteria Grow overnight under standard in vitro culture conditions. Create duplicate plates and arrange Used for gridding or mold making for determination.   B. Preparation of grid   Place each of the insert libraries in place in the high-density array. Each transformant host cell (including inserts, increased by PCR) ) Or with the nucleic acids of the clones on a solid support.   Many conventional methods are used to immobilize clones on the surface of various solid supports. I have. For example, Affinity Techniques, Enzyme Purification: Part P, Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M .; Wilcheck, Acad. Press, NY (1971 ); Immobilized Biochemicals and Affinity Chromatography, Advances inExper Imental Medicine and Biology, Vol. 42, ed. R. Dunlap, Plenum Press, NY (1 974); U.S. Pat. No. 4,762,881; U.S. Pat. No. 4,542,102; EP 391,60. No. 8 (October 10, 1990); or US Pat. No. 4,992,127 (1989 November 21).   One desirable method for anchoring clones to a solid support is described in International Application No.PCT / US 90/06607 (published March 30, 1991). Simply put, this one The method comprises forming a predetermined region capable of immobilizing clones on the surface of a solid support. It includes. This method applies to surfaces that allow selective activation of a given area. The use of a bonded substrate is formed. Upon activation, these binding substrates are It binds to clones derived from the nome library and has immobilization ability.   Nucleotide alignment at predetermined regions on its surface for hybridization. Any known solid substrate suitable for binding a sequence and its nucleotide sequence The method for fixing the can be used by those skilled in the art according to the present invention. Wear. Similarly, to enable detection of hybridization of immobilized clones Known methods such as fluorescence, radioactivity, photoactivation, energy transfer colors Elemental, biotinylation, solid state circuit formation, and the like may be used in the present invention. III. The method of the present invention   The present invention relates to the closure of a gap and subsequent establishment of a computer-generated assembly. For high-throughput sequencing and full-length coding sequences In one embodiment, the composition described above is used.   A. High-throughput sequencing   In the present invention, a small insert library is used for high-throughput nucleotide sequencing. Can be In a preferred embodiment, it is obtained by standard sequencing. The base sequencing reaction is performed up to the application range of 2 to 3 times the range (for example, Flei schmann et al. (See Science, (1995), 269: p.496). The resulting sequence is GELMERGE A available from Genetics Computer Group, Inc. of Madison, WI (UWGCG) Assemble using a standard computer program such as ssembler. The Assembler identifies non-overlapping regions, or gaps, between the yangmburi. These gaps account for incomplete sampling and library-derived clones. The statistical consequences of non-randomness in the collection of sequence fragments due to deletion Arises.   B. 1 Gap closure   In one embodiment of gap closure, the primer pairs are assembled next to each other. Prepared from the non-overlapping ends of the tangent sequence and added to the total genomic DNA isolated from the selected organism For each individual PCR reaction. Assembled three types of probes Can be prepared from known non-overlapping ends of a sequence: (1) including the ends of the assembled sequence M Clones can be labeled (ie clones can be used directly as probes) (2) a primer pair derived from the end of the sequence obtained by assembling the PCR fragment; And (3) an oligonucleotide derived from the end of the assembled sequence. Otides can be used as hybridization probes. these The product of the reaction is preferably directly labeled with a radioisotope or fluorescent label and In separate hybridization reactions on the loaded library Use as lobe.   B. 2 Simple interest of clones containing the full-length coding region   Adjacent sequences were identified by positively hybridizing probes and sequence Confirm by analysis and computer assembly. In addition, two separate gis If the probe from the end of the cap hybridizes to the same gridded clone, In that case, the region spanning the gap is immediately identified. Primer walking the whole claw Until the gap is closed or the gap is closed. New probes and Hybridization is performed as needed.   C. Adjacent sequence assembly or alignment   As will be appreciated by those of skill in the art upon reading this disclosure, It can be placed in approximate order prior to sequencing. For example, in one specific example, 1 Hybridize a 00 kb cosmid insert against a small insert library Rediscover and identify adjacent clones prior to sequencing. More positive of clones Precise alignment is performed with enzymes with few cleavage sites (E. coli NotI, PacI, Spe However, it varies depending on the base composition of the organism. Preferably, the enzyme is Cut once every 10-100 kb. ) Digestion of genomic DNA, or more often Partial digestion with an enzyme that frequently cleaves (preferably once every 10-100 kb) Large DNA fragments obtained by digestion by pulse field gel electrophoresis Separation and isolation of fragments, hybridization to grids of isolated fragments This is performed by dicing.   D. Other methods of the invention   As will be apparent to those of skill in the art upon reading this disclosure, the compositions and methods of the present invention May be used for similar processes. For example, in one embodiment, a large The first pass sequencing of the desert library was performed on the cosmid vector sequence. This is done using both universal forward and reverse primers. m13 array Universal primers, such as primers for etc., are well known in the art. Start a small number of sequencing and use a standard computer such as GELMERGE Assembler Sequence these large insert clones in adjacent sequences using the You. Other computer-assisted assemblies of nucleotide data are It is known in the field. For example, "Automated DNA Sequencing and Ana1ysis", Adams et al. eds., Academic Press (1995), (E.W. Myers presents a discussion of s. oftware systems for fragment assembly in Chapter 32); and J.D.Kececiog lu et al., “Combinatorial Algorithms for DNA Sequence Assembly”, Algori thmica, 13: 7-51 (1995)). Then, the appropriate clone (ie, other To a small insert library grid. Can be selected for use as a lobe. Identify clones with universal primers To determine the nucleotide sequence and assemble the sequence data. In this example, Designing primers for the second walk in the insert library Eliminate the expense and time to build.   The present invention is further described by the following non-limiting examples. Example Example 1   A genomic library of Staphylococcus aureus was used as a vector, Lambda Z Built with APII. The average size of the inserts in this library is about 5k b. Primary sequence analysis using universal forward and reverse primers Performed on approximately 10,000 clones from this library. Then Ann Finalize the preprocessing steps for notating and grouping Functional assembly similar to GELMERGE Assembler except before assembly The derived DNA sequence was assembled using Lie.   Annotating considers two dissimilar sequences to be similar or otherwise May produce incorrect results in the grouping or assembly process Method for identifying partial gene sequences and regions of putative gene assembly. You. These areas are then grouped and assembled in the method of the invention. Seems to hinder the accuracy of the The remaining unidentified regions are grouped and And useful information (for assembly and assembly purposes) Grouping and assembly processes. Can interfere with subsequent steps Regions identified as potential are ignored in these steps.   For example, the region that can be identified in the annotating step is a region other than the target species. Sequences from cell types and from cell structures such as ribosomes and mitochondria It is a nucleic acid or DNA. Polynucleotide runs, simple tandem repeats (STR) and Of low-information regions that occur multiple times in sequences such as genomic repeats and ALUs can do. In addition, ambiguous areas and experimental errors or artificial The resulting region can also be identified.   After annotating, the annotated partial gene sequence is Group with the annotated partial gene sequence. Annotating The step of grouping the partial gene sequences Based on the binding relationship between the sequence and other unannotated partial gene sequences Of other existing annotated partial gene sequences Some may be components of the putative gene assembly identified earlier. This Steps are annotating regions from partial gene sequences and previously identified inferences Start by ignoring gene assembly. Then annotating The partial gene sequence is ignored, ignoring the Is compared with the consensus sequence of the putative gene assembly identified above. Then the partial remains The gene sequences are compared with each other, ignoring the annotated regions. The partial heredity Groups are determined based on the similarities determined by these comparisons in the child sequences. Like this The resulting group contains a set of partial gene sequences that are considered to belong to each other. I.e., the grouping process is likely to be assembled together Child Form a group of arrays.   For each group in the previous step, the positional alignment of the related partial gene sequences Based on the assumption that all subgene sequences belong to the same putative gene assembly. This is done as a group. One of the results of the alignment is one or more putative gene assembly May be the result of the alignment, the alignment step of which is the glue of the partial gene sequence. It may expose inconsistencies between loops.   Once positional alignment has been completed for each putative gene assembly, Prepared by various contig assemblies known to those skilled in the art. For example, GELMER GE (UWGCG, Madison, WI).   Once the unnotating, grouping, and assembly steps are complete, Save the constant gene assembly in a database. Putative gene assembly Analyze based on its sequence, structure, biochemical function or other relevant characteristics Is also good. Once categorized, the database is organized into its potential biochemical functions, structures, Or information related to putative gene assembly for other characteristics .   For example, one method of analyzing putative gene assembly is to analyze for other known genes. By homology. Shared homology of putative gene assembly with known genes May suggest a similar biochemical role or function.   Another typical method for analyzing putative gene assembly is to use the known sequence based on. Specific sequence patterns include signal sequence, transmembrane domain, SH2 domain Encoding protein regions with specific biochemical properties, such as Have been.   After the assembly process, non-overlapping flanking sequence ends were identified. This library The corresponding number of sequenced clones from the To prepare a high-density array.   Two clones, 2AU2142 and 2AU0165, are located at the ends of adjacent sequences. Containing non-overlapping sequences. PCR primers are used for these non-overlapping Designed to amplify a 250 bp fragment for each of the adjacent sequence ends And a separate PCR reaction on the genomic DNA of Staphylococcus aureus. It was used for. The PCR product was purified, radiolabeled and separated into separate Used for the hybridization reaction. PCR for clone 2AU2142 The fragment was found to hybridize to three clones . Restriction digest analysis indicated that these three clones had overlapping sequences. Indicated. Sequence analysis confirmed this result, and further extended from the end of the adjacent sequence. An additional 450 bp was shown. 2 AU0165 derived from the fragment Hybridized to the clone. Restriction fragments and Sequence analysis also confirmed the overlapping nature of these clones.   Analyzing a partial gene sequence without departing from the scope or spirit of the invention It will be apparent to those skilled in the art that various modifications may be made to the methods of the present invention for

Claims (1)

[Claims] 1. High-throughput sequencing of selected organisms, assembly of flanking sequences, The method of closing the system gap is:   (A) preparing a first random genomic library of the selected organism;   (B) sequencing the library of step (a) to identify adjacent nucleotide sequences And;   (C) assembling adjacent sequences and identifying non-overlapping ends or grids;   (D) at the non-overlapping end of the assembled nucleotide sequence from the selected organism Creating a corresponding labeled hybridization probe;   (E) preparing a second random genomic library of the selected organism;   (F) a plurality of regions derived from the genomic library of step (e) Obtaining a grid having a surface to which the limited material is fixed;   (G) hybridizing the probe of step (d) to the grid, non-overlapping Identifying the sequence spanning the termini;   (H) determining the nucleotide sequence of the sequence spanning the non-overlapping ends;   (I) Close gaps between adjacent sequences assembled by sequences spanning non-overlapping ends If not, the length corresponding to the newly identified non-overlapping end identified in step (h) A labeled hybridization probe, and use the additional probe. And repeating the hybridization step (g) for the grid of step (f). And determining the nucleotide sequence of the sequence spanning the non-overlapping ends. 2. The first random genomic library contains a small insert library The method of claim 1. 3. The second random genomic library provides a medium insert library The method of claim 1 comprising: 4. The second random genome library contains a large insert library The method of claim 1. 5. Step (g) is:   (G1) The probe of step (d) is hybridized to a grid, Identifying sequences spanning the overlapping ends;   (G2) detectable sequence identified in step (g1) spanning non-overlapping ends Label;   (G3) the plurality of restricted materials from the genomic library of step (a) Obtaining a grid having a surface that is fixed to a predetermined area of the surface;   (G4) hybridizing the sequence identified in step (g2) to the second grid Rediscover and identify sequences spanning non-overlapping ends Including   Step (h) is:   (H1) Nucleotide of the sequence identified in step (g4) spanning non-overlapping ends Determine the sequence of the sequence;   (H2) Assemble adjacent sequences   The method of claim 1 comprising: 6. The hybridization probe of step (d) has non-overlapping ends 2. The method according to claim 1, comprising material from the genomic library (a). 7. When the hybridization probe in step (d) is non- PCR amplified fragment to the end of assembled sequence with overlapping ends 2. The method of claim 1, wherein the method comprises: 8. The hybridization probe of step (d) has non-overlapping ends 2. The method according to claim 1, comprising an oligonucleotide corresponding to the end of the assembled sequence. Method. 9. An isolated full-length gene sequence identified by the method of claim 1. 10. An isolated protein produced by expression of the gene sequence according to claim 9. quality. 11. 10. The method of claim 9 for use in treating a disease associated with growth of an organism. A therapeutic compound capable of regulating the expression of a gene sequence. 12. 11. The method according to claim 10, for use in the treatment of a disease associated with the growth of an organism. A therapeutic compound capable of modulating the activity of a protein. 13. A disease comprising a reagent capable of detecting the gene sequence according to claim 9 as a target or Is a diagnostic composition useful for diagnosing infection.