Analysis of the Quality and Utility of Random Shotgun Sequencing at Low Redundancies

Abstract

The currently favored approach for sequencing the human genome involves selecting representative large-insert clones (100-200 kb), randomly shearing this DNA to construct shotgun libraries, and then sequencing many different isolates from the library. This method, entitled directed random shotgun sequencing, requires highly redundant sequencing to obtain a complete and accurate finished consensus sequence. Recently it has been suggested that a rapidly generated lower redundancy sequence might be of use to the scientific community. Low-redundancy sequencing has been examined previously using simulated data sets. Here we utilize trace data from a number of projects submitted to GenBank to perform reconstruction experiments that mimic low-redundancy sequencing. These low-redundancy sequences have been examined for the completeness and quality of the consensus product, information content, and usefulness for interspecies comparisons.The data presented here suggest three different sequencing strategies, each with different utilities.(1) Nearly complete sequence data can be obtained by sequencing a random shotgun library at sixfold redundancy. This may therefore represent a good point to switch from a random to directed approach. (2) Sequencing can be performed with as little as twofold redundancy to find most of the information about exons, EST hits, and putative exon similarity matches. (3) To obtain contiguity of coding regions, sequencing at three-to fourfold redundancy would be appropriate. From these results, we suggest that a useful intermediate product for genome sequencing might be obtained by three-to fourfold redundancy. Such a product would allow a large amount of biologically useful data to be extracted while postponing the majority of work involved in producing a high quality consensus sequence.

Sequence and Comparative Analysis of the Mouse 1-Megabase Region Orthologous to the Human 11p15 Imprinted Domain

“…The majority of the mouse sequence analyzed in this study reflects draft sequence assemblies (Collins et al 1998). The value of the draft sequence, which is anticipated to provide >90% coverage (Bouck et al 1998), has been greatly enhanced through the availability of sequence from an orthologous region of a second species.…”

“…Draft assemblies at this level of coverage contain the vast majority of the clone sequence (>90%), with the remaining sequence gaps being small (<1 kb; Bouck et al 1998). Although the outcome of a draft assembly is a series of sequence contigs of unknown order and orientation, sequence alignments to references (other genomic sequences, genes, etc.)…”

“…can be used to determine the correct positioning of the draft sequence contigs. Deeper coverage sequencing ‫)ן21-01(‬ and assembly, especially using paired forward/reverse reads from sequencing subclones, further reduces the gap number and can generate self-ordered contig sets (Bouck et al 1998). For the RP23-92l23 clone, deeper coverage sequencing and finishing was performed.…”

“…Vertical black lines show the position of the remaining gaps within the mouse draft assembly sequences. The sequences within these gaps are expected to be <10% (Bouck et al 1998) of the overall region. Where there is disagreement about nomenclature, exons are numbered arbitrarily (e.g., Igf2).…”

An intermediate grade of finished genomic sequence suitable for comparative analyses

“…First, the shotgun sequence reads must provide sufficient redundancy to ensure a high-accuracy consensus sequence and long-range continuity following assembly; when applied to a diverse set of different species' genomes, this most reliably and cost-effectively can be accomplished with greater than eightfold average coverage, thereby comfortably exceeding established minimum thresholds (Bouck et al 1998). Second, the sequence must be devoid of gross misassemblies and regions of notably poor quality (see Methods for details).…”