Invention and early improvements
Origin
Chromosome jumping (or chromosome hopping) was first described in 1984 byBasic principle and original method
This technique is an extension of "chromosome walking" that allows larger "steps" along the chromosome. If steps of length N kb are desired, very high molecular weight DNA is necessary. Once isolated, it is partially digested with a frequent-cuttingEarly challenges and improvements
The original technique of chromosome jumping was developed in the laboratories of Collins and Weissman at Yale University in New Haven, U.S. and the laboratories of Poustka and Lehrach at the European Molecular Biology Laboratory in Heidelberg, Germany. Collins and Weissman's method described above encountered some early limitations. The main concern was with avoiding non-circularized fragments. Two solutions were suggested: either screening junction fragments with a given probe or adding a second size-selection step after the ligation to separate single circular clones (monomers) from clones ligated to each other (multimers). The authors also suggested that other markers such as the λ cos site or antibiotic resistance genes should be considered (instead of the amber suppressor tRNA gene) to facilitate selection of junction clones. Poustka and Lehrach suggested that full digestion with rare-cutting restrictions enzymes (such as NotI) should be used for the first step of the library construction instead of partial digestion with a frequently cutting restriction enzyme. This would significantly reduce the number of clones from millions to thousands. However, this could create problems with circularizing the DNA fragments since these fragments would be very long, and would also lose the flexibility in choice of end points that one gets in partial digests. One suggestion for overcoming these problems would be to combine the two methods, i.e. to construct a jumping library from DNA fragments digested partially with a commonly cutting restriction enzyme and completely with a rare cutting restriction enzyme and circularizing them into plasmids cleaved with both enzymes. Several of these "combination" libraries were completed in 1986. In 1991, Zabarovsky et al. proposed a new approach for construction of jumping libraries. This approach included the use of two separate λ vectors for library construction, and a partial filling-in reaction that removes the need for a selectable marker. This filling-in reaction worked by destroying the specific cohesive ends (resulting from restriction digests) of the DNA fragments that were nonligated and noncircularized, thus preventing them from cloning into the vectors, in a more energy-efficient and accurate manner. Furthermore, this improved technique required less DNA to start with, and also produced a library that could be transferred into a plasmid form, making it easier to store and replicate. Using this new approach, they successfully constructed a human NotI jumping library from a lymphoblastoid cell line and a human chromosome 3-specific NotI jumping library from a human chromosome 3 and mouse hybrid cell line.Current method
Second-generation or "Next-Gen" (NGS) techniques have evolved radically: the sequencing capacity has increased more than ten thousandfold and the cost has dropped by over one million-fold since 2007(National Human Genome Research Institute). NGS has revolutionized the genetic field in many ways.Library construction
A library is often prepared by random fragmentation of DNA and ligation of common adaptor sequences. However, the generated short reads challenge the identification of structural variants, such as indels, translocations, and duplication. Large regions of simple repeats can further complicate the alignment. Alternatively, a jumping library can be used with NGS for the mapping of structural variation and scaffolding of de novo assemblies. Jumping libraries can be categorized according to the length of the incorporated DNA fragments.Short-jump library
In a short-jump library, 3 kb genomic DNA fragments are ligated with biotinylate ends and circularized. The circular segments are then sheared into small fragments and the biotinylated fragments are selected by affinity assay for paired-end sequencing. There are two issues related to short-jump libraries. First, a read can pass through the biotinylated circularization junction and reduce the effective read length. Second, reads from non-jumped fragments (i.e. fragments without the circularization junction) are sequenced and reduce genomic coverage. It has been reported that non-jumped fragments range from 4% to 13%, depending on the size of selection. The first problem might be solved by shearing circles into a larger size and select for those larger fragments. The second problem can be addressed by using a custom barcoded jumping library.Custom barcoded jumping library
This jumping library uses adaptors containing markers for fragment selection in combination with barcodes for multiplexing. The protocol was developed by Talkowski et al. and based on mate-pair library preparation for SOLiD sequencing. The selected DNA fragment size is 3.5 – 4.5 kb. Two adaptors were involved: one containing an EcoP15I recognition site and an AC overhang; the other containing a GT overhang, a biotinylated thymine, and an oligo barcode. The circularized DNA was digested and the fragments with biotynylated adaptors were selected for (see Figure 3). The EcoP15I recognition site and barcode help to distinguish junction fragments from nonjump fragments. These targeted fragments should contain 25 to 27bp of genomic DNA, the EcoP15I recognition site, the overhang, and the barcode.Long-jump library
This library construction process is similar to that of the short-jump library except that the condition is optimized for longer fragments (5 kb).Fosmid-jump library
This library construction process is also similar to that of short-jump library except that transfection using the E. coli vector is required for amplification of large (40 kb) DNA fragments. In addition, thePaired-end sequencing
The segments resulting from circularization during constructing jumping library are cleaved, and DNA fragments with markers will be enriched and subjected to paired-end sequencing. These DNA fragments are sequenced from both ends and generate pairs of reads. The genomic distance between the reads in each pair is approximately known and used for the assembly process. For example, a DNA clone generated by random fragmentation is about 200 bp, and a read from each end is around 180 bp, overlapping each other. This should be distinguished from mate-pair sequencing, which is basically a combination of next generation sequencing with jumping libraries.Computational analysis
Different assembly tools have been developed to handle jumping library data. One example is DELLY. DELLY was developed to discover genomic structural variants and "integrates short insert paired-ends, long-range mate-pairs and split-read alignments" to detect rearrangements at sequence level. An example of joint development of new experimental design and algorithm development is demonstrated by the ALLPATHS-LG assembler.Confirmation
When used for detection of genetic and genomic changes, jumping clones require validation byApplications
Early applications
In the early days, chromosome walking from genetically linked DNA markers was used to identify and clone disease genes. However, the large molecular distance between known markers and the gene of interest was complicating the cloning process. In 1987, a human chromosome jumping library was constructed to clone the cystic fibrosis gene. Cystic fibrosis is an autosomal recessive disease affecting 1 in 2000 Caucasians. This was the first disease in which the usefulness of the jumping libraries was demonstrated. Met oncogene was a marker tightly linked to the cystic fibrosis gene on human chromosome 7, and the library was screened for a jumping clone starting at this marker. The cystic fibrosis gene was determined to localize 240kb downstream of the met gene. Chromosome jumping helped reduce the mapping "steps" and bypass the highly repetitive regions in the mammalian genome. Chromosome jumping also allowed the production of probes required for faster diagnosis of this and other diseases.New applications
Characterizing chromosomal rearrangements
Balanced chromosomal rearrangements can have a significant contribution to diseases, as demonstrated by the studies of leukemia. However, many of them are undetected by chromosomal microarray.Prenatal diagnosis
Conventional cytogenetic testing cannot offer the gene-level resolution required to predict the outcome of a pregnancy and whole genome deep sequencing is not practical for routine prenatal diagnosis. Whole-genome jumping library could complement conventional prenatal testing. This novel method was successfully applied to identify a case of CHARGE syndrome.De novo assembly
InLimitation
The cost of sequencing has dropped dramatically while the cost of construction of jumping libraries has not. Therefore, as new sequencing technologies and bioinformatic tools are developed, jumping libraries may become redundant.See also
*References
External links