Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.

OBJECTIVE: We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. STUDY DESIGN: Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. RESULTS: In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%). CONCLUSION: Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

Evaluation of linkage disequilibrium between chromosome 22q11 single nucleotide polymorphisms in a large outbred population.

To assess the utility of linkage disequilibrium (LD) as a tool for fine-mapping disease genes in non-isolated populations, we have assessed the linkage disequilibrium strength among a series of single nucleotide polymorphisms (SNPs) in an approximate 1 Mb region of human chromosome 22q11. Nineteen random SNPs were discovered and tested across this region with an average spacing of 57 kb (range=1.4-289 kb). These 19 SNPs were genotyped in a population consisting of 444 unrelated pedigrees that were largely collected in the U.S. and U.K. Haplotypes for all pedigrees were derived from pedigree data and over 1,400 haplotypes from unrelated individuals were evaluated for linkage disequilibrium between marker alleles. In addition, linkage disequilibrium between marker alleles was also evaluated using estimated haplotypes without genealogical information (i.e., without parental genotype information). Every marker pair combination was tested for a total of 171 tests and 2x2 contingency tables were constructed to measure LD strength. In general the haplotypes derived from pedigree data provided a more conservative estimate of LD strength. Using genealogical information for estimates of D', 59% (10/17) of marker pairs less than 50 kb apart had D' values >0.30. Finally, we observed a 60 kb region with non-significant LD, which could reflect increased recombination in this region.

A genomic analysis of the archaeal system Ignicoccus hospitalis-Nanoarchaeum equitans.

BACKGROUND: The relationship between the hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans is the only known example of a specific association between two species of Archaea. Little is known about the mechanisms that enable this relationship. RESULTS: We sequenced the complete genome of I. hospitalis and found it to be the smallest among independent, free-living organisms. A comparative genomic reconstruction suggests that the I. hospitalis lineage has lost most of the genes associated with a heterotrophic metabolism that is characteristic of most of the Crenarchaeota. A streamlined genome is also suggested by a low frequency of paralogs and fragmentation of many operons. However, this process appears to be partially balanced by lateral gene transfer from archaeal and bacterial sources. CONCLUSIONS: A combination of genomic and cellular features suggests highly efficient adaptation to the low energy yield of sulfur-hydrogen respiration and efficient inorganic carbon and nitrogen assimilation. Evidence of lateral gene exchange between N. equitans and I. hospitalis indicates that the relationship has impacted both genomes. This association is the simplest symbiotic system known to date and a unique model for studying mechanisms of interspecific relationships at the genomic and metabolic levels.

Targeted access to the genomes of low-abundance organisms in complex microbial communities.

Current metagenomic approaches to the study of complex microbial consortia provide a glimpse into the community metabolism and occasionally allow genomic assemblies for the most abundant organisms. However, little information is gained for the members of the community present at low frequencies, especially those representing yet-uncultured taxa, which include the bulk of the diversity present in most environments. Here we used phylogenetically directed cell separation by fluorescence in situ hybridization and flow cytometry, followed by amplification and sequencing of a fraction of the genomic DNA of several bacterial cells that belong to the TM7 phylum. Partial genomic assembly allowed, for the first time, a look into the evolution and potential metabolism of a soil representative from this group of organisms for which there are no species in stable laboratory cultures. Genomic reconstruction from targeted cells of uncultured organisms isolated directly from the environment represents a powerful approach to access any specific members of a community and an alternative way to assess the community's metabolic potential.

Identification of rice (Oryza sativa) proteins linked to the cyclin-mediated regulation of the cell cycle.

Yeast two-hybrid assays were used to identify rice proteins interacting with two rice cyclins and other proteins potentially involved in cell cycling. The DNA sequences encoding 119 protein fragments identified were then compared by BLAST against proteins in GenBank. The proteins found include myosin-like proteins, transcription factors, kinesins, centromere proteins and undefined proteins. Based on interactions with cyclins and other elements required for cycling, we believe the undefined proteins may be involved in associated cycling processes. The identification of proteins involved in cell cycle regulation in rice may allow for the control of agronomic traits involving plant growth or development.

A network of rice genes associated with stress response and seed development.

We used a systematic approach to build a network of genes associated with developmental and stress responses in rice by identifying interaction domains for 200 proteins from stressed and developing tissues, by measuring the associated gene expression changes in different tissues exposed to a variety of environmental, biological, and chemical stress treatments, and by localizing the cognate genes to regions of stress-tolerance trait genetic loci. The integrated data set suggests that similar genes respond to environmental cues and stresses, and some may also regulate development. We demonstrate that the data can be used to correctly predict gene function in monocots and dicots. As a result, we have identified five genes that contribute to disease resistance in Arabidopsis.

A high-throughput Arabidopsis reverse genetics system.

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).

The genome of the japonica subspecies of rice, an important cereal and model monocot, was sequenced and assembled by whole-genome shotgun sequencing. The assembled sequence covers 93% of the 420-megabase genome. Gene predictions on the assembled sequence suggest that the genome contains 32,000 to 50,000 genes. Homologs of 98% of the known maize, wheat, and barley proteins are found in rice. Synteny and gene homology between rice and the other cereal genomes are extensive, whereas synteny with Arabidopsis is limited. Assignment of candidate rice orthologs to Arabidopsis genes is possible in many cases. The rice genome sequence provides a foundation for the improvement of cereals, our most important crops.