A BAC-based physical map of the Drosophila buzzatii genome

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps and provide useful resources for sequencing entire genomes. Drosophila buzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and a ~;18X expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be ~;23X. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9,555 clones, and assembled them using FingerPrinted Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670 singletons. Finally, we anchored 181 large contigs (containing 7,788 clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community

A BAC-based physical map of the Drosophila buzzatii genome

Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps and provide useful resources for sequencing entire genomes. Drosophila buzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and a ~;18X expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be ~;23X. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9,555 clones, and assembled them using FingerPrinted Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670 singletons. Finally, we anchored 181 large contigs (containing 7,788 clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community

A BAC clone fingerprinting approach to the detection of human genome rearrangements

We present a method, called fingerprint profiling (FPP), that uses restriction digest fingerprints of bacterial artificial chromosome clones to detect and classify rearrangements in the human genome. The approach uses alignment of experimental fingerprint patterns to in silico digests of the sequence assembly and is capable of detecting micro-deletions (1-5 kb) and balanced rearrangements. Our method has compelling potential for use as a whole-genome method for the identification and characterization of human genome rearrangements

Specificity of individual restriction fragments and patterns based on exact and experimental sizing tolerance

<p><b>Copyright information:</b></p><p>Taken from "A BAC clone fingerprinting approach to the detection of human genome rearrangements"</p><p>http://genomebiology.com/2007/8/10/R224</p><p>Genome Biology 2007;8(10):R224-R224.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2246298.</p><p></p> dIII restriction fragment specificity for the human genome for fragments within the experimental size range of 500 bp to 30 kb. For a given fragment size, the vertical scale represents the fraction of fragments in the genome that are indistinguishable by size in the case of either exact sizing (fragments in common between two fingerprints must be of identical size) or within experimental tolerance (fragments in common between two fingerprints must be within experimental sizing error; Figure 3) on a fingerprinting gel. When sizing is exact, fragment specificity follows approximately the exponential distribution of fragment sizes and spans a range of 3.5 orders of magnitude. When experimental tolerance is included, the number of distinguishable fragment size bins is reduced and the range of fragment specificity drops to two orders of magnitude. The specificity of a fingerprint pattern of a given size in the human genome. Fingerprint pattern size is measured in terms of number of fragments. Regions with identical patterns are those in which there is a 1:1 mapping within tolerance between all sizeable fragments. The specificity of experimental fingerprint patterns is cumulatively affected by specificity of individual fragments. The specificity of fragments is sufficiently low (that is, due to high experimental precision) so that 96.5% of the genome is uniquely represented by fragment patterns of 8 fragments or more

Detailed reconciliation of sequence and fingerprint alignments for clone 3F05, which contains at least four internal breakpoints

<p><b>Copyright information:</b></p><p>Taken from "A BAC clone fingerprinting approach to the detection of human genome rearrangements"</p><p>http://genomebiology.com/2007/8/10/R224</p><p>Genome Biology 2007;8(10):R224-R224.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2246298.</p><p></p> FPP is capable of dissecting complex rearrangements in a clone, as illustrated in this figure showing the internal structure of M0003F05. This BAC was sequenced [26] and found to be composed of content from at least five distinct regions (A-E). FPP detected 4/5 of these regions. BLAT (grey rectangles with alignment orientation arrows) and FPP (thin black lines) alignments of M0003F05 are shown; values underneath coordinate pairs are differences in edge positions between BLAT and FPP alignments

Simulation results of sensitivity and spatial error of rearrangement detection by FPP using experimental sizing tolerance

<p><b>Copyright information:</b></p><p>Taken from "A BAC clone fingerprinting approach to the detection of human genome rearrangements"</p><p>http://genomebiology.com/2007/8/10/R224</p><p>Genome Biology 2007;8(10):R224-R224.</p><p>Published online 22 Oct 2007</p><p>PMCID:PMC2246298.</p><p></p> Sensitivity is measured as the fraction of clone regions of a given size with successful FPP alignments and is plotted for five digests (labeled 1-5). Spatial error is measured by the median distance between FPP and theoretical alignment positions. The largest improvement in both sensitivity and spatial error is realized by migrating FPP from one digest to two. With two fingerprint patterns used to align the clone, 50% of >25 kb clone regions are aligned (90% of >45 kb regions) with a spatial error of 1.7 kb

A set of BAC clones spanning the human genome

Using the human bacterial artificial chromosome (BAC) fingerprint-based physical map, genome sequence assembly and BAC end sequences, we have generated a fingerprint-validated set of 32 855 BAC clones spanning the human genome. The clone set provides coverage for at least 98% of the human fingerprint map, 99% of the current assembled sequence and has an effective resolving power of 79 kb. We have made the clone set publicly available, anticipating that it will generally facilitate FISH or array-CGH-based identification and characterization of chromosomal alterations relevant to disease

Integrated and Sequence-Ordered BAC- and YAC-Based Physical Maps for the Rat Genome

As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide ∼13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome