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1.  A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA 
PLoS ONE  2013;8(10):e76096.
DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8–11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.
doi:10.1371/journal.pone.0076096
PMCID: PMC3810253  PMID: 24204593
2.  Reduced dosage of ERF causes complex craniosynostosis in humans and mice, and links ERK1/2 signaling to regulation of osteogenesis 
Nature genetics  2013;45(3):308-313.
The extracellular signal-related kinases (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets1. Here we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs 2-7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf reduced to ~30% of normal exhibit postnatal multisuture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to distal regulatory elements containing RUNX or AP1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.
doi:10.1038/ng.2539
PMCID: PMC3683605  PMID: 23354439
3.  Discovering the RNA Transcription Landscape using Directional Approaches 
High-throughput complementary DNA sequencing (RNA-Seq) is a powerful technique that allows for sensitive digital quantification of transcript levels. Moreover, RNA-Seq enables the detection of non-canonical transcription start sites and termination sites, alternative splice isoforms and transcript mutation and edition. Standard “next-generation” RNA-sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information. In this approach, the synthesis of randomly primed double-stranded cDNA followed by addition of adaptors for sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Our objective was to address this need by developing a novel streamlined, low input method for Directional RNA-Sequencing that highly retains strand orientation information while maintaining even coverage of transcript expression. This method is based on second strand labeling and excision after adaptor ligation; allowing differential tagging of the first strand cDNA ends. As a result, we have enabled strand specific mRNA sequencing, as well as whole transcriptome sequencing (Total RNA-Seq) from ribosomal-depleted samples. Total RNA-Seq provides a much broader picture of expression dynamics including discovery of antisense transcripts. This work presents a streamlined, fast solution for complete RNA sequencing, with high quality data that illustrates the complexity and diversity of the RNA transcription landscape.
PMCID: PMC3635307
4.  A Fast Solution to NGS Library Prep with Low Nanogram DNA Input 
Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.
PMCID: PMC3635320
6.  A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis 
Nucleic Acids Research  2000;28(5):1067-1077.
A robust Tn7-based in vitro transposition system is described that displays little target site selectivity, allowing the efficient recovery of many different transposon insertions in target DNAs ranging from small plasmids to cosmids to whole genomes. Two miniTn7 derivatives are described that are useful for the analysis of genes: one a derivative for making translational and transcriptional target gene fusions and the other a derivative that can generate 15 bp (5 amino acid) insertions in target DNAs (proteins).
PMCID: PMC102592  PMID: 10666445

Results 1-6 (6)