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1.  Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment 
PLoS ONE  2014;9(5):e96492.
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. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA.
PMCID: PMC4008621  PMID: 24788618
2.  MicroRNAs and their isomiRs function cooperatively to target common biological pathways 
Genome Biology  2011;12(12):R126.
Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules.
To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs.
Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.
PMCID: PMC3334621  PMID: 22208850
3.  Development of Personalized Tumor Biomarkers Using Massively Parallel Sequencing 
Science translational medicine  2010;2(20):20ra14.
Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. The evaluation of patient-specific translocations in leukemias and lymphomas has revolutionized diagnostics for these diseases. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers with massively parallel sequencing revealed an average of nine rearranged sequences (range, 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints was able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.
PMCID: PMC2858564  PMID: 20371490
4.  A small cell lung cancer genome reports complex tobacco exposure signatures 
Nature  2009;463(7278):184-190.
Cancer is driven by mutation. Worldwide, tobacco smoking is the major lifestyle exposure that causes cancer, exerting carcinogenicity through >60 chemicals that bind and mutate DNA. Using massively parallel sequencing technology, we sequenced a small cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. 22,910 somatic substitutions were identified, including 132 in coding exons. Multiple mutation signatures testify to the cocktail of carcinogens in tobacco smoke and their proclivities for particular bases and surrounding sequence context. Effects of transcription-coupled repair and a second, more general expression-linked repair pathway were evident. We identified a tandem duplication that duplicates exons 3-8 of CHD7 in-frame, and another two lines carrying PVT1-CHD7 fusion genes, suggesting that CHD7 may be recurrently rearranged in this disease. These findings illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer.
PMCID: PMC2880489  PMID: 20016488
5.  Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding 
PLoS ONE  2010;5(2):e9320.
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. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.
PMCID: PMC2825269  PMID: 20179767
6.  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.
PMCID: PMC2777346  PMID: 19949697
SNP; massively parallel sequencing; hybridization; high density oligonucleotide array
7.  Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChIP-seq 
Genome Biology  2009;10(11):R129.
FOXA1 and FOXA3 binding patterns in HepG2 cells, together with their possible molecular interactions with FOXA2 and each other, are revealed by ChIP-seq.
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.
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. Using co-immunoprecipitation, we further show that FOXA2 interacts with both FOXA1 and FOXA3 in vivo, while FOXA1 and FOXA3 do not appear to interact. Additionally, we detected diverse patterns of trimethylation of lysine 4 on histone H3 (H3K4me3) at transcriptional start sites and directionality of this modification at FOXA binding sites. Using the sequence reads at polymorphic positions, we were able to predict allele specific binding for FOXA1, FOXA3, and H3K4me3. Finally, several SNPs associated with diseases and quantitative traits were located in the enriched regions.
We find that ChIP-seq can be used not only to create gene regulatory maps but also to predict molecular interactions and to inform on the mechanisms for common quantitative variation.
PMCID: PMC3091322  PMID: 19919681
8.  ALLPATHS 2: small genomes assembled accurately and with high continuity from short paired reads 
Genome Biology  2009;10(10):R103.
Allpaths2, a method for accurately assembling small genomes with high continuity using short paired reads.
We demonstrate that genome sequences approaching finished quality can be generated from short paired reads. Using 36 base (fragment) and 26 base (jumping) reads from five microbial genomes of varied GC composition and sizes up to 40 Mb, ALLPATHS2 generated assemblies with long, accurate contigs and scaffolds. Velvet and EULER-SR were less accurate. For example, for Escherichia coli, the fraction of 10-kb stretches that were perfect was 99.8% (ALLPATHS2), 68.7% (Velvet), and 42.1% (EULER-SR).
PMCID: PMC2784318  PMID: 19796385
9.  Mapping and sequencing of structural variation from eight human genomes 
Nature  2008;453(7191):56-64.
Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale—particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation—a standard for genotyping platforms and a prelude to future individual genome sequencing projects.
PMCID: PMC2424287  PMID: 18451855
10.  Protein interaction mapping on a functional shotgun sequence of Rickettsia sibirica 
Nucleic Acids Research  2004;32(3):1059-1064.
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. We apply the approach by shotgun sequencing and annotating the genome of Rickettsia sibirica strain 246, an obligate intracellular human pathogen among the Spotted Fever Group rickettsiae. The bacteria invade endothelial cells and cause lysis after large amounts of progeny have accumulated. Little is known about specific Rickettsial virulence factors and their mode of pathogenicity. Analysis of the combined genomic sequence and protein–protein interaction data for a set of virulence related Type IV secretion system (T4SS) proteins revealed over 250 interactions and will provide insight into the mechanism of Rickettsial pathogenicity.
PMCID: PMC373392  PMID: 14872061

Results 1-10 (10)