Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

We have developed a new approach to screen bacterial artificial chromosome (BAC) libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380) with temperature inducible homologous recombination (HR) capability. We amplified one library segment, induced HR at 42°C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies

Energy spread of ultracold electron bunches extracted from a laser cooled gas

Ultrashort and ultracold electron bunches created by near-threshold femtosecond photoionization of a laser-cooled gas hold great promise for single-shot ultrafast diffraction experiments. In previous publications the transverse beam quality and the bunch length have been determined. Here the longitudinal energy spread of the generated bunches is measured for the first time, using a specially developed Wien filter. The Wien filter has been calibrated by determining the average deflection of the electron bunch as a function of magnetic field. The measured relative energy spread $\frac{\sigma_{U}}{U} = 0.64 \pm 0.09\%$ agrees well with the theoretical model which states that it is governed by the width of the ionization laser and the acceleration length

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

A physical map of the chicken genome

Strategies for assembling large, complex genomes have evolved to include a combination of whole-genome shotgun sequencing and hierarchal map-assisted sequencing. Whole-genome maps of all types can aid genome assemblies, generally starting with low-resolution cytogenetic maps and ending with the highest resolution of sequence. Fingerprint clone maps are based upon complete restriction enzyme digests of clones representative of the target genome, and ultimately comprise a near-contiguous path of clones across the genome. Such clone-based maps are used to validate sequence assembly order, supply long-range linking information for assembled sequences, anchor sequences to the genetic map and provide templates for closing gaps. Fingerprint maps are also a critical resource for subsequent functional genomic studies, because they provide a redundant and ordered sampling of the genome with clones. In an accompanying paper we describe the draft genome sequence of the chicken, Gallus gallus, the first species sequenced that is both a model organism and a global food source. Here we present a clone-based physical map of the chicken genome at 20-fold coverage, containing 260 contigs of overlapping clones. This map represents approximately 91% of the chicken genome and enables identification of chicken clones aligned to positions in other sequenced genomes

Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing

Background RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. Conclusions These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.Medicine, Faculty ofScience, Faculty ofNon UBCMedical Genetics, Department ofMicrobiology and Immunology, Department ofReviewedFacult

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