Showing papers by "Timothy B. Stockwell published in 2008"

Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome.

Abstract: We have synthesized a 582,970 bp Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted “watermarks” at intergenic sites known to tolerate transposon insertions. Overlapping “cassettes” of 5 to 7 kb, assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb (“1/8 genome”), and 144 kb (“1/4 genome”), which were all cloned as bacterial artificial chromosomes (BACs) in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformationassociated recombination (TAR) cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments. M. genitalium is a bacterium with the smallest genome of any independently replicating cell that has been grown in pure

Genetic variation in an individual human exome.

Abstract: There is much interest in characterizing the variation in a human individual, because this may elucidate what contributes significantly to a person's phenotype, thereby enabling personalized genomics. We focus here on the variants in a person's ‘exome,’ which is the set of exons in a genome, because the exome is believed to harbor much of the functional variation. We provide an analysis of the ∼12,500 variants that affect the protein coding portion of an individual's genome. We identified ∼10,400 nonsynonymous single nucleotide polymorphisms (nsSNPs) in this individual, of which ∼15–20% are rare in the human population. We predict ∼1,500 nsSNPs affect protein function and these tend be heterozygous, rare, or novel. Of the ∼700 coding indels, approximately half tend to have lengths that are a multiple of three, which causes insertions/deletions of amino acids in the corresponding protein, rather than introducing frameshifts. Coding indels also occur frequently at the termini of genes, so even if an indel causes a frameshift, an alternative start or stop site in the gene can still be used to make a functional protein. In summary, we reduced the set of ∼12,500 nonsilent coding variants by ∼8-fold to a set of variants that are most likely to have major effects on their proteins' functions. This is our first glimpse of an individual's exome and a snapshot of the current state of personalized genomics. The majority of coding variants in this individual are common and appear to be functionally neutral. Our results also indicate that some variants can be used to improve the current NCBI human reference genome. As more genomes are sequenced, many rare variants and non-SNP variants will be discovered. We present an approach to analyze the coding variation in humans by proposing multiple bioinformatic methods to hone in on possible functional variation.

Novel computational methods for increasing PCR primer design effectiveness in directed sequencing

Abstract: Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research. The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.