Intraspecific phylogenetic analysis of Siberian woolly mammoths using complete mitochondrial genomes.

We report five new complete mitochondrial DNA (mtDNA) genomes of Siberian woolly mammoth (Mammuthus primigenius), sequenced with up to 73-fold coverage from DNA extracted from hair shaft material. Three of the sequences present the first complete mtDNA genomes of mammoth clade II. Analysis of these and 13 recently published mtDNA genomes demonstrates the existence of two apparently sympatric mtDNA clades that exhibit high interclade divergence. The analytical power afforded by the analysis of the complete mtDNA genomes reveals a surprisingly ancient coalescence age of the two clades, approximately 1-2 million years, depending on the calibration technique. Furthermore, statistical analysis of the temporal distribution of the (14)C ages of these and previously identified members of the two mammoth clades suggests that clade II went extinct before clade I. Modeling of protein structures failed to indicate any important functional difference between genomes belonging to the two clades, suggesting that the loss of clade II more likely is due to genetic drift than a selective sweep.

Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes.

The paired-end ditagging (PET) technique has been shown to be efficient and accurate for large-scale transcriptome and genome analysis. However, as with other DNA tag-based sequencing strategies, it is constrained by the current efficiency of Sanger technology. A recently developed multiplex sequencing method (454-sequencing) using picolitre-scale reactions has achieved a remarkable advance in efficiency, but suffers from short-read lengths, and a lack of paired-end information. To further enhance the efficiency of PET analysis and at the same time overcome the drawbacks of the new sequencing method, we coupled multiplex sequencing with paired-end ditagging (MS-PET) using modified PET procedures to simultaneously sequence 200,000 to 300,000 dimerized PET (diPET) templates, with an output of nearly half-a-million PET sequences in a single 4 h machine run. We demonstrate the utility and robustness of MS-PET by analyzing the transcriptome of human breast carcinoma cells, and by mapping p53 binding sites in the genome of human colorectal carcinoma cells. This combined sequencing strategy achieved an approximate 100-fold efficiency increase over the current standard for PET analysis, and furthermore enables the short-read-length multiplex sequencing procedure to acquire paired-end information from large DNA fragments.

Expression profiling using a hexamer-based universal microarray.

We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific knowledge of transcript sequences. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (approximately 80-120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (4(6)) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. The technology was also used to demonstrate expression profiling from a hybrid organism in a proof-of-concept experiment where a T-cell receptor gene was expressed in yeast.

Whole-genome shotgun sequencing of mitochondria from ancient hair shafts.

Although the application of sequencing-by-synthesis techniques to DNA extracted from bones has revolutionized the study of ancient DNA, it has been plagued by large fractions of contaminating environmental DNA. The genetic analyses of hair shafts could be a solution: We present 10 previously unexamined Siberian mammoth (Mammuthus primigenius) mitochondrial genomes, sequenced with up to 48-fold coverage. The observed levels of damage-derived sequencing errors were lower than those observed in previously published frozen bone samples, even though one of the specimens was >50,000 14C years old and another had been stored for 200 years at room temperature. The method therefore sets the stage for molecular-genetic analysis of museum collections.

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety.

BACKGROUND: Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. PRINCIPAL FINDINGS: We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). CONCLUSIONS: Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

Unique features of a highly pathogenic Campylobacter jejuni strain.

Campylobacter jejuni, a major human enteric pathogen, exhibits significant strain-to-strain differences which result in differences in pathogenic potential. C. jejuni 81-176 is a highly virulent strain that exhibits unique pathogenic features and is used by many research laboratories. We have determined the nucleotide sequence of its genome and compared it to the genomes of other sequenced C. jejuni strains. We identified a number of unique genetic features which may confer specific metabolic and pathogenic properties on this strain. We have also identified regions of the C. jejuni genome that are hot spots for the integration of horizontally acquired genetic material. This information should help the understanding of the pathogenesis of C. jejuni and, in particular, the unique features of this highly pathogenic strain.

Comparison of two CYP2D6 genotyping methods and assessment of genotype-phenotype relationships.

BACKGROUND: There have been no published reports comparing the CYP450 GeneChip microarray assay with more standard methods of genetic testing. METHODS: We collected 20-mL blood samples from 236 volunteers for DNA isolation and testing before each individual ingested 60 mg of dextromethorphan, and collected their urine. CYP2D6 alleles *3 to *7, *9, *17, and *41, and multiple CYP2D6 gene copies were tested by allele-specific PCR (AS-PCR), whereas alleles *2 to *4 and *6 to *11 were tested by the Affymetrix CYP450 GeneChip assay. Five of the CYP2D6 alleles (*3, *4, *6, *7, and *9) were tested by both AS-PCR and the CYP450 GeneChip assay in an independent and blinded fashion in 232 of the 236 healthy volunteers. The combined CYP2D6 genotype from both methods was used to divide the population into four subgroups, poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs), and ultrarapid metabolizers (UMs), based on their relative function and ability to express the CYP2D6 gene. The urinary elimination of dextromethorphan was assessed in each of these CYP2D6 subgroups. RESULTS: The CYP2D6*3, *4, *6, *7, and *9 alleles showed a high degree of concordance between the CYP450 GeneChip and AS-PCR methods (>99% concordance). The mean (SD) of the log[dextromethorphan metabolic ratio (MR)] in the four CYP2D6 subgroups was PM = 0.49 (0.38); IM = -1.24 (0.53); EM = -2.35 (0.61); and UM = -2.43 (0.38). CONCLUSIONS: Oligonucleotide microarray technology is an efficient and reliable way to test for CYP2D6 gene variation based on five alleles compared by separate methods. The methodology is influenced by the quality and amount of DNA present. The log(dextromethorphan MR) is a highly variable index that appears to reflect the crude nature of the dextromethorphan MR as an indicator of CYP2D6 in vivo enzyme activity.