Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.

Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.

We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in mammalian cells. By obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

Genome sequence, comparative analysis and haplotype structure of the domestic dog.

Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.

Transition of a traditional pharmacology course for dental students to an online delivery format: a pilot project.

The purpose of this report is to describe the rationale and subsequent transition of a pharmacology course for dental students from a traditional face-to-face lecture format to online delivery using a course management system (CMS). A dental school faculty member with dental and pharmacology degrees and a Ph.D. was asked to serve as course director and to develop and implement a nontraditional course using the Blackboard CMS technology, which houses asynchronous course content materials, study guides, and online resource materials. Respondus software was used to create, manage, and administer weekly online quizzes. A comprehensive midterm and final examination were conducted in a traditional face-to-face setting. A survey was used to capture student satisfaction with this self-directed introductory pharmacology course. Participants were second-year dental students (Classes of 2011 and 2012). There was a survey response rate of 91 percent (179/197). The Likert-style survey questions produced ordinal data from which the median and interquartile range were calculated. On a scale in which 1=Poor, 5=Excellent, the median evaluation for the instructor was 4 (IQR=1.5). On a global question that asked how students rate the course overall, the median score was 4 (IQR=1.0). Results show that a majority of students were positive about the online delivery of the introductory pharmacology course and for many students this was their first online course experience. Resistance to self-directed learning was a theme with those students who rated the course poorly. In a comparison of overall course grades from the previous year, student performance in this course was much stronger. As a result of student feedback seeking more interaction with the course director, it was determined that the next time the course is offered there will be additional opportunities for greater face-to-face time with the instructor. Ongoing evaluation will be important as new teaching technologies emerge and are adopted for teaching and learning.

A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454.

We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.

Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.

Targeting genomic loci by massively parallel sequencing requires new methods to enrich templates to be sequenced. We developed a capture method that uses biotinylated RNA 'baits' to fish targets out of a 'pond' of DNA fragments. The RNA is transcribed from PCR-amplified oligodeoxynucleotides originally synthesized on a microarray, generating sufficient bait for multiple captures at concentrations high enough to drive the hybridization. We tested this method with 170-mer baits that target >15,000 coding exons (2.5 Mb) and four regions (1.7 Mb total) using Illumina sequencing as read-out. About 90% of uniquely aligning bases fell on or near bait sequence; up to 50% lay on exons proper. The uniformity was such that approximately 60% of target bases in the exonic 'catch', and approximately 80% in the regional catch, had at least half the mean coverage. One lane of Illumina sequence was sufficient to call high-confidence genotypes for 89% of the targeted exon space.

Quality scores and SNP detection in sequencing-by-synthesis systems.

Promising new sequencing technologies, based on sequencing-by-synthesis (SBS), are starting to deliver large amounts of DNA sequence at very low cost. Polymorphism detection is a key application. We describe general methods for improved quality scores and accurate automated polymorphism detection, and apply them to data from the Roche (454) Genome Sequencer 20. We assess our methods using known-truth data sets, which is critical to the validity of the assessments. We developed informative, base-by-base error predictors for this sequencer and used a variant of the phred binning algorithm to combine them into a single empirically derived quality score. These quality scores are more useful than those produced by the system software: They both better predict actual error rates and identify many more high-quality bases. We developed a SNP detection method, with variants for low coverage, high coverage, and PCR amplicon applications, and evaluated it on known-truth data sets. We demonstrate good specificity in single reads, and excellent specificity (no false positives in 215 kb of genome) in high-coverage data.