The chloroplast genome hidden in plain sight, open access publishing and anti-fragile distributed data sources.

We sequenced several cannabis genomes in 2011 of June and the first and the longest contigs to emerge were the chloroplast and mitochondrial genomes. Having been a contributor to the Human Genome Project and an eye-witness to the real benefits of immediate data release, I have first hand experience with the potential mal-investment of millions of dollars of tax payer money narrowly averted due to the adopted global rapid data release policy. The policy was vital in reducing duplication of effort and economic waste.

Hurt, tired and queasy: Specific variants in the ATPase domain of the TRAP1 mitochondrial chaperone are associated with common, chronic "functional" symptomatology including pain, fatigue and gastrointestinal dysmotility.

Functional disorders are common conditions with a substantial impact on a patients' wellbeing, and can be diagnostically elusive. There are bidirectional associations between functional disorders and mitochondrial dysfunction. In this study, provided clinical information and the exon sequence of the TRAP1 mitochondrial chaperone were retrospectively reviewed with a focus on the functional categories of chronic pain, fatigue and gastrointestinal dysmotility.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase.

Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment.

We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment.

An integrated semiconductor device enabling non-optical genome sequencing.

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip.

Whole methylome analysis by ultra-deep sequencing using two-base encoding.

Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites.

Polymorphism discovery in high-throughput resequenced microarray-enriched human genomic loci.

Identifying genetic variants and mutations that underlie human diseases requires development of robust, cost-effective tools for routine resequencing of regions of interest in the human genome. Here, we demonstrate that coupling Applied Biosystems SOLiD system-sequencing platform with microarray capture of targeted regions provides an efficient and robust method for high-coverage resequencing and polymorphism discovery in human protein-coding exons.

Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChIP-seq.

Background: The forkhead box/winged helix family members FOXA1, FOXA2, and FOXA3 are of high importance in development and specification of the hepatic linage and the continued expression of liver-specific genes.

Results: Here, we present a genome-wide location analysis of FOXA1 and FOXA3 binding sites in HepG2 cells through chromatin immunoprecipitation with detection by sequencing (ChIP-seq) studies and compare these with our previous results on FOXA2. We found that these factors often bind close to each other in different combinations and consecutive immunoprecipitation of chromatin for one and then a second factor (ChIP-reChIP) shows that this occurs in the same cell and on the same DNA molecule, suggestive of molecular interactions.

Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage.

Rapid whole-genome mutational profiling using next-generation sequencing technologies.

Forward genetic mutational studies, adaptive evolution, and phenotypic screening are powerful tools for creating new variant organisms with desirable traits. However, mutations generated in the process cannot be easily identified with traditional genetic tools. We show that new high-throughput, massively parallel sequencing technologies can completely and accurately characterize a mutant genome relative to a previously sequenced parental (reference) strain.

Stem cell transcriptome profiling via massive-scale mRNA sequencing.

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology.

Sample preparation.

Today's human genetic studies are requiring an ever-increasing number of sample manipulations. Although a significant investment of time and money are required, automation can considerably reduce human error and improve sample tracking fidelity when applied to such repetitive projects. This unit presents issues involved in the automation of sample preparation for genomic DNA isolation and PCR.

Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype.

Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish.

Protein interaction mapping on a functional shotgun sequence of Rickettsia sibirica.

Protein interaction maps can reveal novel pathways and functional complexes, allowing 'guilt by association' annotation of uncharacterized proteins. To address the need for large-scale protein interaction analyses, a bacterial two-hybrid system was coupled with a whole genome shotgun sequencing approach for microbial genome analysis. We report the first large-scale proteomics study using this system, integrating de novo genome sequencing with functional interaction mapping and annotation in a high-throughput format.

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes.