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We describe a sensitive mRNA profiling technology, PMAGE (for “polony multiplex analysis of gene expression”), which detects messenger RNAs (mRNAs) as rare as one transcript per three cells. PMAGE incorporates an improved ligation-based method to sequence 14-nucleotide tags derived from individual mRNA molecules. One sequence tag from each mRNA molecule is amplified onto a separate 1-micrometer bead, denoted as a polymerase colony or polony, and about 5 million polonies are arrayed in a flow cell for parallel sequencing. Using PMAGE, we identified early transcriptional changes that preceded pathological manifestations of hypertrophic cardiomyopathy in mice carrying a disease-causing mutation. PMAGE provided a comprehensive profile of cardiac mRNAs, including low-abundance mRNAs encoding signaling molecules and transcription factors that are likely to participate in disease pathogenesis.

We report a method for multilocus long-range haplotyping on human chromosome molecules in vitro based on the DNA polymerase colony (polony) technology. By immobilizing thousands of intact chromosome molecules within a polyacrylamide gel on a microscope slide and performing multiple amplifications from single molecules, we determined long-range haplotypes spanning a 153-Mb region of human chromosome 7 and found evidence of rare mitotic recombination events in human lymphocytes. Furthermore, the parallel nature of DNA polony technology allows efficient haplotyping on pooled DNAs from a population on one slide, with a throughput three orders of magnitudes higher than current molecular haplotyping methods. Linkage disequilibrium statistics established by our pooled DNA haplotyping method are more accurate than statistically inferred haplotypes. This haplotyping method is well suited for candidate gene-based association studies as well as for investigating the pattern of recombination in mammalian cells.

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ~1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ~98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. Fifty-one of 74 participants had positive enzyme results (46 carriers, five late-onset Tay-Sachs [LOTS]), 16 had negative, and seven had inconclusive results. NGS + 7.6 kb del screening of HEXA found a pathogenic mutation, pseudoallele, or variant of unknown significance (VUS) in 100% of the enzyme-positive or obligate carrier/enzyme-inconclusive samples. NGS detected the B1 allele in two enzyme-negative obligate carriers. Our data indicate that NGS can be used as a TSD clinical carrier screening tool. We demonstrate that NGS can be superior in detecting TSD carriers compared to traditional enzyme and genotyping methodologies, which are limited by false-positive and false-negative results and ethnically focused, limited mutation panels, respectively, but is not ready for sole use due to lack of information regarding some VUS.

Purpose: Carrier screening for recessive Mendelian disorders traditionally employs focused genotyping to interrogate limited sets of mutations most prevalent in specific ethnic groups. We sought to develop a next-generation DNA sequencing–based workflow to enable analysis of a more comprehensive set of disease-causing mutations. Methods: We utilized molecular inversion probes to capture the protein-coding regions of 15 genes from genomic DNA isolated from whole blood and sequenced those regions using the Illumina HiSeq 2000 (Illumina, San Diego, CA). To assess the quality of the resulting data, we measured both the fraction of the targeted region yielding high-quality genotype calls, and the sensitivity and specificity of those calls by comparison with conventional Sanger sequencing across hundreds of samples. Finally, to improve the overall accuracy for detecting insertions and deletions, we introduce a novel assembly-based approach that substantially increases sensitivity without reducing specificity. Results: We generated high-quality sequence for at least 99.8% of targeted base pairs in samples derived from blood and achieved high concordance with Sanger sequencing (sensitivity >99.9%, specificity >99.999%). Our novel algorithm is capable of detecting insertions and deletions inaccessible by current methods. Conclusion: Our next-generation DNA sequencing–based approach yields the accuracy and completeness necessary for a carrier screening test.

The process of DNA sequencing has made tremendous strides in throughput, improved accuracy, ease of production, and lowered cost. As the practice of DNA sequencing has improved, so has the downstream data analysis with sophisticated databases and bioinformatics tools. Together, these advances have enlarged the number of applications upon which DNA sequencing can be brought to bear. This introductory unit provides a description of DNA sequencing with a focus on current and “NextGen” (second and third generation) automated technologies and applications. Curr. Protoc. Mol. Biol. 96:7.0.1-7.0.18. © 2011 by John Wiley & Sons, Inc. Keywords: NextGen DNA sequencing; high-throughput sequencing; epigenomics; transcriptome; ChIP Seq; CNV; copy number variation

Efficient and cost-effective DNA sequencing technologies are critical to the progress of molecular biology. This overview of DNA sequencing strategies provides a high-level review of seven distinct approaches to DNA sequencing: (a) dideoxy sequencing; (b) solid phase sequencing; (c) sequencing-by-hybridization; (d) mass spectrometry; (e) cyclic array sequencing; (f) microelectrophoresis; and (g) nanopore sequencing. Other platforms currently in development are also briefly described. The primary focus here is on Sanger dideoxy sequencing, which has been the dominant technology since 1977, and on cyclic array strategies, for which several competitive implementations have been developed since 2005. Because the field of DNA sequencing is changing rapidly, this unit represents a snapshot as of September, 2011.

We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation.

Efficient and cost-effective DNA sequencing technologies have been, and may continue to be, critical to the progress of molecular biology. This overview of DNA sequencing strategies provides a high-level review of six distinct approaches to DNA sequencing: (a) dideoxy sequencing; (b) cyclic array sequencing; (c) sequencing-by-hybridization; (d) microelectrophoresis; (e) mass spectrometry; and (f) nanopore sequencing. The primary focus is on dideoxy sequencing, which has been the dominant technology since 1977, and on cyclic array strategies, for which several competitive implementations have been developed since 2005. Because the field of DNA sequencing is changing rapidly, this unit represents a snapshot of this particular moment.

Polony DNA sequencing provides an inexpensive, accurate, high-throughput way to resequence genomes of interest by comparison to a reference genome. Mate-paired in vitro shotgun genomic libraries are produced and clonally amplified on microbeads by emulsion PCR. These serve as templates for sequencing by fluorescent nonamer ligation reactions on a microscope slide. Each sequencing run results in millions of 26-bp reads that can be aligned to the reference genome, allowing the identification of differences between sequences.