Large-scale functional annotation and expanded implementations of the P{wHy} hybrid transposon in the Drosophila melanogaster genome.

Whole genome sequencing of the model organisms has created increased demand for efficient tools to facilitate the genome annotation efforts. Accordingly, we report the further implementations and analyses stemming from our publicly available P{wHy} library for Drosophila melanogaster. A two-step regime-large scale transposon mutagenesis followed by hobo-induced nested deletions-allows mutation saturation and provides significant enhancements to existing genomic coverage. We previously showed that, for a given starting insert, deletion saturation is readily obtained over a 60-kb interval; here, we perform a breakdown analysis of efficiency to identify rate-limiting steps in the process. Transrecombination, the hobo-induced recombination between two P{wHy} half molecules, was shown to further expand the P{wHy} mutational range, pointing to a potent, iterative process of transrecombination-reconstitution-transrecombination for alternating between very large and very fine-grained deletions in a self-contained manner. A number of strains also showed partial or complete repression of P{wHy} markers, depending on chromosome location, whereby asymmetric marker silencing allowed continuous phenotypic detection, indicating that P{wHy}-based saturational mutagenesis should be useful for the study of heterochromatin/positional effects.

Universal Fast Walking for direct and versatile determination of flanking sequence.

We report a highly compact system for accelerating direct genome walking. Unlike previous walking techniques, our strategy does not rely on restriction enzymes or ligases, and is therefore unaffected by the availability of useful restriction sites in the flanking region. A complete circumvention of molecular cloning steps qualifies this method for sequencing genome segments that are regarded unclonable, and thus unsequenceable by the traditional methods. A premium was placed on economy of design: the system comprises just four direct reagent additions, in microliter-scale volumes, over the course of a 6-h procedure. The walk range in this method is directly related to the capabilities of the associated polymerase blend, indicating that it can achieve in excess of 35 kilobases per reaction. It also produces a DNA fingerprint that is distinctive to the flanking sequence. Despite the complexity of banding patterns in these fingerprints, we observed that the reaction products were directly sequenceable. In view of its speed, reliability and generality, we term the described method Universal Fast Walking.

A modified universal fast walking method for single-tube transposon mapping.

An enhanced universal fast walking (UFW) method adapted for the mapping of transposons is described. This protocol combines the original UFW method with the use of agarase to unravel composite nucleotide sequence, thereby forgoing molecular cloning steps and the use of restriction enzymes and ligases necessary in other available genome walking methods such as the prominent inverse PCR. The minuscule automatable chemistry of UFW is completed within one reaction vessel using a constant enzyme buffer, and the intrinsic DNA fingerprints, from which amplicons may be quantitatively recovered, offer quality assurance. The core steps of the protocol, spanning half a day or less, comprise first-strand synthesis, primer destruction, random-ended-primer annealing, distal branched-end repair, second-primer destruction, lariat formation and final amplification. Distinctively, no starting or intermediate templates are wasted during the reaction series, thus achieving yields comparable to direct PCR. Ultimate per-reaction walk-lengths are schematically illimitable and sequence-ready amplicons can be produced immediately from prevalent single-copy genomic walk origins. The core UFW protocol may be applied, as described here, to expedited transposon boundary retrieval, but is also applicable to general genome walking and cDNA walking, as well as viral and other insertional element mapping.

Revisiting the protein-coding gene catalog of Drosophila melanogaster using 12 fly genomes.

The availability of sequenced genomes from 12 Drosophila species has enabled the use of comparative genomics for the systematic discovery of functional elements conserved within this genus. We have developed quantitative metrics for the evolutionary signatures specific to protein-coding regions and applied them genome-wide, resulting in 1193 candidate new protein-coding exons in the D. melanogaster genome. We have reviewed these predictions by manual curation and validated a subset by directed cDNA screening and sequencing, revealing both new genes and new alternative splice forms of known genes. We also used these evolutionary signatures to evaluate existing gene annotations, resulting in the validation of 87% of genes lacking descriptive names and identifying 414 poorly conserved genes that are likely to be spurious predictions, noncoding, or species-specific genes. Furthermore, our methods suggest a variety of refinements to hundreds of existing gene models, such as modifications to translation start codons and exon splice boundaries. Finally, we performed directed genome-wide searches for unusual protein-coding structures, discovering 149 possible examples of stop codon readthrough, 125 new candidate ORFs of polycistronic mRNAs, and several candidate translational frameshifts. These results affect >10% of annotated fly genes and demonstrate the power of comparative genomics to enhance our understanding of genome organization, even in a model organism as intensively studied as Drosophila melanogaster.

A deletion-generator compound element allows deletion saturation analysis for genomewide phenotypic annotation.

With the available eukaryotic genome sequences, there are predictions of thousands of previously uncharacterized genes without known function or available mutational variant. Thus, there is an urgent need for efficient genetic tools for genomewide phenotypic analysis. Here we describe such a tool: a deletion-generator technology that exploits properties of a double transposable element to produce molecularly defined deletions at high density and with high efficiency. This double element, called P[wHy], is composed of a "deleter" element hobo, bracketed by two genetic markers and inserted into a "carrier" P element. We have used this P[wHy] element in Drosophila melanogaster to generate sets of nested deletions of sufficient coverage to discriminate among every transcription unit within 60 kb of the starting insertion site. Because these two types of mobile elements, carrier and deleter, can be found in other species, our strategy should be applicable to phenotypic analysis in a variety of model organisms.