This approach is similar to the clone by clone approach but involves sequencing the ends of more than half a million bacs bacterial arti. Computational bac clone contig assembly for comprehensive. Contigs built with fingerprints, markers, and fpc v4. Yacs in obtaining highquality dna from clones representing the ush1c critical region has resulted in the identification of several ests or potential candidate genes by largescale sequencing and computer analysis. The genome is first broken up into large fragments that are cloned to give a library of overlapping pieces. An obvious solution is to sequence as far as you can from your primer. Actual start of this clone is at base position 1 of dj438o4. Background current whole genome sequencing wgs technologies can provide genotype information of the human genome. Ratings 80% 10 8 out of 10 people found this document helpful. Which of the following is done in clone contig method of. Dna gel reading data obtained from the shotgun method of dna sequencing.
A contig is a set of gel readings that are related to one another by overlap of their sequences. Following electrophoresis and staining of the gel with a sensitive intercalating dye, fingerprints were manually. The process would begin by breaking the genome into bacsized pieces, arranging these bacs into a map, then breaking each bac up into a series of smaller clones, which were usually then also mapped. Obviously, we need to break the genome into smaller fragments. The pac clone contig presented here should greatly facilitate the identification of the gene associated with ush1c. This strategy involves designing universal probes from conserved sequences and utilizing them for hybridizationbased screening of arrayed bac libraries. Research open access on the design of clonebased haplotyping. With such a clone based contig contiguous sequence at hand, the assembly of. May 19, 2011 one of the aims of this bac clone sequencing project was the rapid identification of genes in contig sequences.
An alternative to the clonebyclone method is whole genome shotgun wgs. Which of the following is done in clone contig method of dna sequencing but not in shotgun method of genome sequencing. The map consists of 298 bac contigs and covers 419 mb of the 430mb rice genome. The second method used to evaluate contig reliability involved. Dna probes are directly hybridized both to restriction enzymedigested pools of bac dna, and to dot blots, yielding both qualitative dot and quantitative. Create the following reaction setup, noting that the molar amount of plasmid can be calculated with the. Pdf while the sequencing of bacterial genomes has become a. May 27, 2019 the key difference between clone by clone sequencing and shotgun sequencing lies in their method of conduct. Apr 01, 2000 the bacrf method exploits the ease of purifying genomic dna cloned into bacs to merge highresolution mapping with sequenceready contig assembly in highly duplicated genomes. Contig describes a set of adjacent dna sequences or clones assembled using overlapping sequences or restriction fragments. Using large cloned contigs was the approach that was taken by the official government sponsored human genome project. Automated dna sequencing yields sequences of 500 to. Paired end readsthe forward and reverseprimed sequence reads that define clone ends and insert size.
Because the pieces are assembled into a continuous whole, it makes sense that contig is derived from the word contiguous. Discrepancies in the alignment resulting from gaps and. Contig assembly of bacterial artificial chromosome clones. It is named by analogy with the rapidly expanding, quasirandom shot grouping of a shotgun the chaintermination method of dna sequencing sanger sequencing can only be used for short dna strands of 100 to base pairs. Assembly and analysis of extended human genomic contig regions. If a clone in a contig was not a questionable clone, but had 15 extra bands encountered only in a few cases in this study, the best match for that clone was determined, and the clone was removed, reorganized into another contig, or rearranged within the same contig as necessary. With the emergence of cheaper sequencing and more sophisticated computer programs, researchers have therefore relied on whole genome shotgun sequencing to tackle. These fragments are inserted into highcapacity vectors such as yacs or bacs, which may carry several hundred kb of dna see ch. In bottomup sequencing projects, a contig refers to overlapping sequence data. Our results provide practical guidelines for the development and design of clone based methods to achieve long range, highresolution and accurate haplotypes.
Contig a set of gel readings that are related to one another by overlap of their. Request pdf computational bac clone contig assembly for comprehensive genome analysis comparative genomic hybridization cgh has proved to be a powerful tool for the detection of genome copy. In this protocol, first a physical map is constructed, then clones in the mtp are sequenced one by one 1. Assembly of chromosomescale contigs by efficiently resolving. Contig maps and genomic sequencing identify candidate. The observed patterns of allelic loss define a minimum consensus region of deletion between d10s221 and d10s610. A contig map shows the regions of a chromosome where contiguous dna segments overlap. Once the overlap between the clones has been found and their order relative to the genome known, a scaffold of a minimal subset of these contigs that covers the entire genome is shotgunsequenced.
The redgreen hybrid in the center is the original dsdna to be sequenced. A contig is a set of overlapping dna segments that together represent a consensus region of dna. Locusspecific contig assembly in highlyduplicated genomes. Whole genome shotgun sequencing uses a fraction of the dna that clone by clone sequencing needs. After preparing individual yac dna in agarose by the lyticaselithium dodecyl sulfate method anand et al. Mauve contig mover provides a new method for proposing the relative order of contigs that make up a draft genome based on comparison to a complete or draft reference genome. The functions at the bottom suggest clones to add or bury, and performs the edit if requested. The distance between contigs can be inferred from the mate pair p. Whole genome shotgun sequencing is particularly efficient if there is an existing reference sequence.
The shotgun part comes from the way the clone is prepared for sequencing. Clone by clone strategy a clone by clone strategy involves the initial creation of a physical map of largeinsert clones bacs or fosmids physical maps are built by generating restriction digests of individual clones typically 15x coverage, then using computerbased algorithms to assemble contigs of related clones. A physical map, including a bacpac clone contig, of the williamsbeuren syndromedeletion region at 7q11. Contig formation is a necessary step in determining the sequence of an entire genome by the clone contig sequencing method. Resultsanchoring the clone contigs to a chromosomal location involved the following steps.
Clone contig sequencing method definition of clone. In this paper, a method was described for the parallel construction of targeted, orthologous, sequenceready bac contig maps from multiple species. Chapter 3 bacterial clone contig on xq22 wellcome sanger. Quick notes on genomes genetics biology discussion. Prior to the development of methods for largescale sequencing, comparative mapping represented the primary approach for comparing the genomes of divergent species. The idea is to pepper the dna with sequence reads such that they overlap, and yield, when assembled, the complete sequence of the clone. Pdf whole genome shotgun sequencing guided by bioinformatics. Physical mapping of cloned sequences was once considered a prerequisite for genome sequencing. The clones are then sequenced from both ends using the chain termination method. Given that the length of a single, individual sequencing read is somewhere between 45bp and 700bp, we are faced with a problem determining the sequence of longer fragments, such as the chromosomes in an entire genome of humans 3 x10 9 bp.
These are usually generated by partial restriction see partial digestion of the genome, and cloned into large insert cloning vectors, such as bacs, yacs or cosmids. Nachimuthu saraswathy, ponnusamy ramalingam, in concepts and techniques in genomics and proteomics, 2011. Clone by clone sequencing method involves mapping of chromosomes and cloning prior to sequencing while clone by shotgun sequencing omits both chromosome mapping and cloning steps during the sequencing. Clone manager quick reference getting started for windows to install an individual or workgroup copy, insert the program cd in drive and allow disk to autorun or select start, settings, control panel, and then click addremove programs. Fpc uses clone coordinates in the contig to select the subset of clones that cover the entire contig. What types of data do we want to remember and analyze.
To accelerate rice and other grass species genome research, we developed a genomewide bacbased map of the rice genome. The contig analysis window is initiated from a contig display. Oxford dictionary of biochemistry and molecular biology. Mapping an endometrial cancer tumor suppressor gene at 10q25. Doubleclick the clone manager icon to start the program. Contig maps and genomic sequencing identify candidate genes. By removing the mapping stages, whole genome shotgun sequencing is a much faster process than clone by clone sequencing.
Packaged into a transgenic mice are helpful in the. Difference between clone by clone sequencing and shotgun. Due to this size limit, longer sequences are subdivided into smaller fragments that. It is necessary to construct 24 contig maps, one each for the 22 autosomes, an x and a y chromosome. High throughput sequencing method that employs automated sequencing of random dna fragments automated dna sequencing yields sequences of 500 to bp in length to determine longer sequences you obtain fragmentary sequences and then join them together by overlapping overlapping is an alignment problem, but. On this data we also compared our method to an approach which uses the number of supporting clones to determine contigs.
A clone contig consists of a group of cloned copied pieces of dna representing overlapping regions of a particular chromosome. A library is constructed by cloning large a few hundred kb to a few mb, overlapping dna inserts spanning the entire genome. If clone c a has exactly or approximately the same bands as clone c b,c. Contig assembly dna sequencing gene quantification. All gel readings belong to one and only one contig, and each contig contains at least one gel reading. Clone contig sequencing method definition of clone contig. Green1,2,3 1genome technology branch, national human genome research institute, and 2national institutes of health nih. To resolve all ambiguities and unmapped contigs, further mapping of clones to the consensus linkage map and the integration of the physical map with other maps are necessary. Also known as the clone by clone strategy the mapbased method map first, sequence later topdown sequencing human genome project adopted a mapbased strategy start with welldefined physical map produce shortest tiling path for largeinsert clones assemble the sequence for each clone then assemble the entire.
Contigs can thus refer both to overlapping dna sequence and to overlapping physical segments contained in clones depending on the context. Clonebyclone sequencing was the preferred method during the human genome project. This plasmid carries genes for resistances to several antibiotics. Baylor college of medicine ac002523 begining of sequence overlaps with af007262, end of sequence overlaps with af011889. One standard lowresolution method for finding the physical position of a cloned fragment is insitu hybridization on. It begins with the construction of genomic libraries of restriction fragments covering all the genomic dna or genomic clones of an organism. Cellular dna extracted, broken and cloned to create genomic dna library. A sequenceready bacterial clone contig and a longrange restriction map for a 1mb interval spanning the deletion region were developed as the first step in experiments directed toward the discovery the 10q25 tumor suppressor. The length column shows the total length of contigs in bands and the avemge length of clone in bands since the coulson et al. Figure 2 pac clone contig of the ush1c region generated by segmap using sts content mapping and yac derived alu pcr and pac end probe hybridization data. This method of genomic mapping is called restriction fingerprinting because it identifies a set of restriction sites contained in each clone. In genetics, shotgun sequencing is a method used for sequencing random dna strands. For example, a clone can be compared against the clones in the contig or against the rest of the database. Aug 01, 2001 genomewide physical mapping with bacteriabased largeinsert clones e.
The inserts in various randomly chosen plasmids were then sequenced to give the smaller fragments shown. Clone contig snp 1 sts 2 bac reference population number of animals method restriction enzyme chromosome position cm ctg1. The clone contig approach is the conventional method for obtaining the sequence of a eukaryotic genome and has also been used with those microbial genomes. The clone by clone sequencing method has been used to sequence several genomes including a.
It was broken up, and smaller pieces cloned into plasmids. Following electrophoresis and staining of the gel with a. Parallel construction of orthologous sequenceready clone. Then, in section 3, we formulate the contig scaffolding problem, and show that the associated decision problem is npcomplete. A physical map, including a bacpac clone contig, of the. To sequence a clone longer than the average read length, it is possible to use a shotgun approach. Parallel construction of orthologous sequenceready clone contig maps in multiple species james w. In total, 86 of 90 snp markers were added to the linkage map see table 1, anchoring 56 different clone contigs and. Ta cloning method 10 protocol clone gene fragment into vector 5. As a result clone by clone sequencing, although reliable and methodical, takes a very long time. Clone, contigs, genome analysis, annotation introduction.
The process of clone by clone shotgun sequencing can be conceptually divided into a series of discrete and. The word contig is derived from the word contiguous. The length of a clone in a contig is equal to the number of its bands, and the overlap between the coordinates of the two clones is approximately the number of shared bands. The individual clones are then contiguated for production of the final sequence. When raw shotgun sequence data from an automated sequencer is fed to this assembler program, it first identifies and trims the vector sequences and also generates the low quality 5 and 3 traces. False mapping is more detrimental than ambiguity since it is not detected during contig mapping. Clone by clone sequencing and clone by shotgun sequencing are two methods of modernday genome sequencing. In our previous work, we applied a combination of experimental and computational method by using fosmid clone sequencing to assist the. Contig mapping is a technique which relies on the use of overlapping clones, referred to as contigs. A clone contig map contig is a short form of contiguous comprises a set of ordered occurring consecutively partially overlapping clones comprising all the dna of an individual chromosome without any gaps. Clone by clone strategy a clone by clone strategy involves the initial creation of a physical map of largeinsert clones bacs or fosmids physical maps are built by generating restriction digests of individual clones typically 15x coverage, then using computerbased. Which of the following is done in clone contig method of dna. Large, mammalian genomes are particularly difficult to clone, sequence and assemble because of their size and structural complexity.
Clone digests were loaded on agarose gels according to their position in the contig to facilitate comparison of fingerprints among related clones fig. Sanger and ngs methods are applied to the sequencing of. Annotating the genome sequence and creating a physical map. To sequence a clone larger than the average read length, it is possible to use a shotgun approach. Nov 30, 2010 ltc differs from fpc in a number of important features that are. A sequence contig is an extended sequence created by merging primary sequences that overlap. As part of an effort to clone the epm1 gene on the basis of its chromosomal location, we have constructed a 753kb bacterial clone contig that encompasses the region containing the gene.
Identification of dna or more specialized cloning vector or the vectors. Our approach yielded a basically correct contig set. Pdf chromosomal assignment of chicken clone contigs by. In section 4, we discuss some simple strategies that one can employ to avoid problems caused by polymorphisms, repeats and misassembled contigs. State the strengths and limitations of the shotgun, wholegenome shotgun and clone contig methods of genome sequencing. Dish detergent and into cloning notes pdf file will let us produce a specific use of open up the simplest method used for creating a restriction enzymes and their genome.
Since the number of barley flcdnas available for this purpose 5,006 is limited compared to the total number of barley genes, the efficiency of barley gene identification was estimated based on homologous rice genes. Mapping an endometrial cancer tumor suppressor gene at. Highresolution physical mapping by construction of contig maps of overlapping clones to determine the positions of genomic landmarks with much greater resolution, we can replace the chromosomes themselves with twentyfour contig maps, one for each of our twentytwo homologous chromosome pairs and one for each of our two sex chromosomes. To assess clone integrity and confirm overlap, a hindiii fingerprint was obtained for all of the clones in the contig.
Another approach, used to sequence very large amounts of dna such as an entire genome. Wholegenome sequencing and assembly with highthroughput. The greedy pathmerging algorithm for contig scaffolding 605 table i. Using the clone contigs, we restrict the search for overlapping reads to a. A novel application of the mauve aligner and viewer provides an automated reordering algorithm coupled with a powerful drilldown display allowing detailed. The level of reliability is determined by the reliability of the methods used to.
Each contig is a genomic clone, usually in a cosmid or a yac. In order to make it easier to talk about our data gained by the shotgun method of sequencing we have invented the word contig. The functions at the top evaluate the current contig. Assembly and analysis of extended human genomic contig. Pdf construction of a 750kb bacterial clone contig and.
Highresolution physical mapping by construction of contig maps of overlapping clones. Bacterial artificial chromosomebased physical map of the. The greedy pathmerging algorithm for contig scaffolding. High throughput sequencing method that employs automated sequencing of random dna fragments. The ability to compare genomes in a reliable fashion. Geneticists use following two methods for sequencing the genomes. Physical mapping federation of american scientists. The idea is to pepper the dna with sequence reads such that they overlap, and when assembled, yield the complete sequence of the clone.
995 1542 66 67 140 228 713 1327 225 1291 1514 639 393 673 731 1097 1487 495 453 734 1193