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  1. Identification and Biosynthesis of Thymidine Hypermodifications in the Genomic DNA of Widespread Bacterial Viruses (2018)

    Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their geneLc material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the DelPia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α- putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2ʹ-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635–7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy) methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.

  2. Kinetic Comparison of Cas9 Homologs Recognizing Diverse PAM Sequences (2018)

    Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share about 17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics we directly compare SpyCas9 and SauCas9 activities. In contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.

  3. AGBT 2018 Wellcome Trust Sanger Institute poster: Thurston. S., et al. (2018). A Large Genome Centre Core Pipeline Refresh.


  4. Amplification Reagents for Molecular Diagnostics Applications (2017)

    Molecular diagnostic methods to detect DNA and RNA targets represent a significant and growing focus for point-of-care and rapid testing. Though varied in application, design, and even mechanism, these methods all rely on enzymes to carry out the reactions. NEB has long provided reagents and enzymes to enable molecular biology research, including providing reliable and novel materials for molecular diagnostic applications. Isothermal amplification methods have emerged as a promising option for reliable point-of-care diagnostics and our research and development on these techniques have produced novel and more versatile isothermal DNA polymerases for faster and robust amplification; WarmStart® technology to permit room-temperature reaction set-up and consistent performance; and colorimetric LAMP detection for field and point-of-care applications. For customization of methods and use, reagents are tested for stability in glycerol-free formats and compatibility with lyophilization. And to enable a wide range of intended applications, we have developed reagents that support the next generation of tests in isothermal (SDA, NASBA, LAMP) and PCR or qPCR methods. Developing better reagents for molecular diagnostics is essential to broader adoption and reach of these powerful tools.

  5. Examining Sources of Error in PCR by Single-Molecule Sequencing (2017)

    The Polymerase Chain Reaction (PCR) is an integral part of many NGS sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. We utilized a single-molecule sequencing assay to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage caused by thermocycling.

  6. Colorimetric LAMP: Visual Detection for Simple Diagnostics (2017)

    Much of modern biology and medicine depends on the ability to accurately determine the identity of a target, whether virus, tumor, mystery infection, or even an uncertain type of food. Fortunately, all living things and viruses carry unique identifiers in their DNA or RNA genomes. Once a sequence identifier is determined, we still need to be able to find it, using a nucleic acid amplification method for precisely identifying a particular sequence. This amplification can be analyzed to see what is made and how much of a particular DNA was present in the sample to begin with. And only the specific target of interest will be amplified, enabling accurate detection of the sequences that we are looking for. With demands and applications for nucleic acid identification growing all the time, new methods have been developed to allow detection more easily, rapidly, and in more settings. A popular example is loop-mediated isothermal amplification (LAMP) which works at a single temperature (isothermal) using DNA polymerases with a special ability to go through double-stranded DNA without heating (“strand displacement”) activity. NEB developed a novel colorimetric format that reacts to DNA synthesis by changing color from pink to yellow as a direct visual response to DNA polymerase adding bases to the growing DNA products. This simple readout of amplification, paired with the speed and robustness of LAMP make for a useful diagnostic tool, with LAMP being used for easy detection of targets everywhere from farms to doctors’ offices, and recently, the International Space Station! If it has DNA or RNA, we can find it, and LAMP will let it be done easily, rapidly and right where you need the answer. While there are plenty of those places on Earth, we’ll soon have them in space too, and with methods like LAMP our astronauts, their food supplies, and their homes can be kept safe.

  7. High Quality Automation Libraries Prepared from a Broad Range of DNA Input Amounts (2017)

    Next Generation Sequencing (NGS) is expanding its applications. Laboratories are increasingly implementing NGS and incrementing the number of samples to process. The ability to construct high quality sequencing libraries in a fast turnaround time has become critical. Automating sequencing library preparation reduces bottlenecks, enables higher throughput and minimizes human errors. This work describes the flexible, automated NEBNext® Ultra™ II DNA library preparation protocols on the TECAN® Freedom EVO® NGS workstation. This simple workflow allows for highly reproducible library preparation from a wide range of DNA input (picograms to a microgram), and with variable quality (from intact to heavily degraded FFPE samples). Automation of this protocol on the Freedom EVO NGS workstation enables flexible sample numbers from 1-96, and minimizes hands-on time with minimal user intervention. High input (200ng) and low input (500pg) human and yeast genomic DNA libraries generated on the Freedom EVO NGS workstation have comparable library performance (high yield, absence of adaptor dimer) to those obtained from manual libraries. NEXTSeq® sequencing data shows high quality libraries. The high percentage of aligned reads (>97.7% mapped reads and >99.03% mapped in pairs) and the low percentage of chimeras (<1%) and adaptor-mapping reads (<0.001%) observed indicate that the Tecan automation of the NEBNext Ultra II DNA Library Prep workflow enables high quality sequence data, even with very low input amounts. GC coverage information obtained indicates that automated Ultra II DNA libraries have very uniform coverage across the range of GC content. This automated method provides a much-needed resource for the reliable preparation of DNASeq libraries from a broad range of sample types and input amounts.

  8. A monoclonal antibody for transcriptome-wide N6-methyladenosine analysis (2017)

    N6-methyladenosine (m6A) has been shown to be the most common base modification in eukaryotic messenger RNA (mRNA) other than the 7-methylguanosine cap. Recent m6A-RNA immunoprecipitation (m6A-RIP) with polyclonal antibodies combined with RNA high-throughput sequencing (RNA-seq) studies have identified the location of m6A sites in a transcriptome-wide manner in a variety of tissues and have started to analyze the function of m6A in mRNA (1-8). In humans m6A is most commonly associated with a sequence motif in the 3’ UTR of mRNAs near stop codons and m6A modification is dependent upon a complex consisting of the methyltranferases METTL3+METTL14 and accessory proteins such as WTAP and KIAA1429.

    To further advance our understanding of m6A in RNA, it is important to continue improving the tools needed for m6A research. Here we present the generation of a new m6A-specific rabbit monoclonal antibody and its use in m6A-RIP-seq experiments.

  9. Single-Strand Specific, Plasmid Borne DNA Methyltransferases M.BceJIII and M.EcoGIX Regulate Plasmid and Single-Stranded Phage Replication. (2017)

    The role of prokaryotic DNA methyltransferases within restriction-modification systems has been well established, while the functional role of the many orphan DNA methyltransferases is still far from clear. Two examples of Dam- and Dcm- orphan DNA methyltransferases have been extensively studied and functional roles in mismatch DNA repair, DNA replication and phase variation of protein expression have been established.   
    We took advantage of the recently developed platform for single-molecule real time sequencing by Pacific Biosciences to investigate DNA methyltransferase specificity.  The analysis of total DNA from two pathogenic strains of B. cenocepacia J2315 and E.coli O104:H4 genomic DNA has revealed the presence of two unusual methyltransferases not previously characterized. Both are plasmid-encoded by ORFs in pBCA072 for B. cenocepacia J2315 and pESBL for E.coli O104:H4.  They both result in single-stranded, almost non-specific m6A modification, within the motif SAB (where S = C or G and B = C, G or T). This methylation is partial and only detected on plasmid DNA.  We have called these enzymes M.BceJIII and M.EcoGIX respectively. 

    A set of genetic and biochemical experiments suggested that the activity of these enzymes is associated with plasmid replication and depended on the origin of replication. While ColEI and p15 origins support plasmid modification, the pSC101 origin does not.  Moreover, we demonstrated that these enzymes work as a complex with DNA polymerase I during plasmid replication and may modify the lagging strand. It is possible they control plasmid and phage replication by discriminating DNA polymerase I-dependent and non-dependent plasmids origins. We suggest that the base flipping inherent to DNA modification may allow the methylase to perform a DNA helicase function and thereby help to control the rate of DNA polymerization to prevent excessive recombination.


  10. Complete Genome Sequence and Methylome Analysis of Beggiatoa leptomitoformis strains D401 and D402 (2016)

    The taxonomy of Beggiatoa genus is still a work in progress. Despite many morphotypes of the Beggiatoa genus having been described in the literature, only one species, B. alba has been validated until now. In 2016, we described a second species, B. leptomitoformis. Two strains of B. leptomitoformis D401 and D402 have been isolated from different regions of Russia. They differ in their morphology and physiology especially their ability to growth lithotrophically in the presence of thiosulfate.  While B. leptomitoformis D402 is able to accumulate elemental sulfur, strainD401 cannot.  We performed genomic sequencing of these two strains using the PacBio SMRT platform and assembled the reads into two complete circular genomes: B. leptomitiformis D-401 with 4,266,286 bp and D-402 with 4,265,296 bp. Both genome sequences have been deposited in GenBank with accession numbers CP018889 and CP012373 respectively (1). Surprisingly these two genomes showed almost 99% identity. The preliminary analysis of several thiosulfate oxidation (,soxA, soxF and soxW) and autotrophic  assimilation of CO2 (cbbQ, rbcL) metabolic operones in both stains did not revel any noticeable differences.

    The advantage of the PacBio sequencing platform is its ability to detect the epigenetic state of the sequenced DNA, which allows for the identification of modified nucleotides and the corresponding motifs in which they occur. Thirteen DNA methyltransferase recognition motifs were found. They include one m4C and nine m6A modifications that were detected by direct SMRT sequencing and an additional three m5C motifs were detected in Tet2 treated DNA. The motifs were then matched with methyltransferase genes in the genome, and the results have been deposited in REBASE (2).

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