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  1. Application of a Novel, Targeted Sequencing-Based Genotyping Approach for Cost Effective Marker Assessment in O. sativa (2019)

    Decreases in sequencing costs have increased the availability of public SNP databases while necessitating development of targeted genotyping assays for use in marker assisted genomic selection for a variety of crop species. The NEBNext Direct Genotyping Solution is a novel, hybridization-based target enrichment approach that has been optimized for use in genotyping applications to increase the number of assays that can be performed in a single reaction, while providing sequencing coverage depth suitable for SNP identification. The approach enables high-levels of multiplexing of both isolates and markers, allowing enrichment of hundreds of thousands of SNP targets in a single hybridization reaction, and the protocol is easily completed in a single day.

    We developed a panel covering the 1,996 single nucleotide polymorphisms previously identified as markers for polymorphism detection in O. Sativa. Here, we demonstrate the application of this panel to cost-effectively enrich defined SNP markers in a highly specific and uniform manner prior to next-generation sequencing.

  2. NEBNext® Ultra™II FS DNA: A Robust Enzyme-based DNA Library Preparation Method Compatible with Plant and Animal Samples (2019)

    Next generation sequencing (NGS) is currently an important tool used in many fields to answer biological questions. DNA fragmentation is the critical initial step in the construction of high quality NGS libraries, however, current fragmentation methods create a bottleneck in library preparation throughput. To meet this challenge, we have developed a robust library construction method (NEBNext Ultra II FS) that integrates enzyme-based DNA fragmentation with end-repair and dA-tailing in a single step, followed by adaptor ligation in the same tube. This method eliminates the need for expensive equipment to fragment DNA; moreover, the optimized workflow reduces the numerous cleanup and liquid transfer steps, reducing the time, cost, and errors associated with library construction.

    The robustness of the Ultra II FS DNA library preparation workflow was tested using genomic DNA from a variety of sources including the model organism Arabidopsis thaliana, the less- documented genome of Cannabis sativa, and Sus scrofa (pig). Libraries were prepared from a range of DNA inputs to achieve different insert sizes with or without PCR amplification. All libraries were sequenced, reads aligned to the appropriate reference genome, and quality metrics generated using Picard tools. Compared with the traditional, mechanical shearing based library preparation method, Ultra II FS is significantly easier to automate, has higher library conversion rate and similar or superior sequencing quality. We further discuss several applications of Ultra II FS in plant and animal research, including genome assembly and sample quality control.

  3. Highly Multiplexed, Targeted Sequencing for Genotyping Maize with the NEBNext Direct Approach (2019)

    argeted next-generation sequencing of molecular markers is a desirable approach to genotype crops for marker-assisted breeding. These methods offer several advantages over other genotyping approaches, including the ability to interrogate thousands of variant sites with a single assay while providing additional information on nearby sequences. However, NGS-based genotyping is typically more expensive than traditional genotyping methods, and for marker-assisted selection, many samples need to be screened to identify individuals to cross. Thus, it is important that the approach used to prepare samples for sequencing is high-throughput and that the cost per sample is low. Here we present the NEBNext Direct Genotyping Solution, a novel, capture-by-hybridization method that allows for processing of up to 9216 samples in a single 96-well plate.

    To demonstrate the capabilities of this approach, we applied the NEBNext Direct Genotyping Solution to genotype maize genomic DNA. We developed a panel of over 4600 legacy SNPs from the Panzea project and tested the ability of the panel to evenly enrich targets from 25 ng of maize DNA. Additionally, because the baits were individually synthesized, subsets of the panel could be rapidly generated to reduce sequencing costs when fewer targets were required. To demonstrate this ability, we selected a 100 marker subpool from the larger bait set and observed consistent coverage of the selected targets while maintaining the high specificity and uniformity of the panel. With this one day, highly multiplexed protocol, hundreds of samples could be processed in a high-throughput manner, making this approach ideal for genomic selection in maize.

  4. High-Throughput Screening of 2300 Genetic Markers in S.lycopersicum using the NEBNext Direct Multiplexed Genotyping Approach (2019)

    Targeted DNA sequencing is rapidly being adopted for the molecular screening of markers during selective crop breeding. For these applications, the need for cost-effective and high-throughput technologies to process large numbers of samples is imperative. Here we describe a novel capture- by-hybridization method for targeted genotyping of crops. This simple workflow allows processing of up to 9216 samples in a single 96-well plate in one day and is easily automated.

    The NEBNext Direct Genotyping Solution can target 100 to 5000 markers from up to 96 samples within a single hybridization. Here we developed a panel targeting 2300 SNPs in the tomato crop, Solanum lycopersicum. Baits were placed within 75 nucleotides of the targeted SNPs, allowing for an efficient sequencing run of 75 bases of target sequencing, 8 bases of sample barcode, 8 bases of hybridization barcode, and 12 bases of a unique molecular identifier (UMI) for filtering PCR duplicates. After an initial screening of the panel, the bait concentrations were adjusted by performance to ensure uniform coverage of the targets. The optimized panel resulted in greater than 90% of the sequencing reads mapping to targeted regions and highly uniform coverage. As a result, this approach reduced the cost and increased the throughput of crop sequencing while generating robust data to reliably genotype multiple varieties of S. lycopersicum.

  5. Uncovering the Cannabis sativa methylome through enzymatic methyl-seq (2019)

    Cannabis sativa is an industrial crop producing more economic value than the top five crops in California combined. In the medical field, cannabinoids are used to treat and alleviate symptoms for a wide range of diseases, including nausea in cancer patients undergoing chemotherapy. Sex determination in cannabis is an important economic factor. Pollination dramatically reduces cannabinoid expression in female plants. To express high levels of cannabinoids, female plants are grown in isolation of male pollen. However, female plants are capable of stress induced hermaphroditism that can lead to acres of pollination and low cannabinoid yields.

    5-methylcytosine (5mC) is an important epigenetic mechanism that regulates many cellular processes, including sex determination. To better understand the hermaphroditic process in cannabis, more robust 5mC surveying tools are required. The current gold standard for determining 5mC, whole genome bisulfite sequencing (WGBS), introduces DNA damage and GC-biased sequences resulting in skewed methylation profiles. To further complicate matters, the cannabis genome is 66% AT-rich and 83% AT-rich after bisulfite conversion. Very few technologies currently address the complexity of plant methylation signals, and to date no methylome exists for cannabis.

    NEBNext Enzymatic Methyl-seq (EM-seqTM) is a novel enzymatic method used to detect 5mC. EM-seq successfully identified the 5mCs within the cannabis genome. Libraries were generated using genomic DNA extracted from female leaf for EM-seq and WGBS. To give insight into the methylation-based regulation of the hermaphroditic process, EM-seq libraries were also prepared using genomic DNA from female flowers, female seeded flowers, and male flowers. We conclude that EM-seq is superior to WGBS and delivers higher library yields, more accurate methylation information, reduced DNA damage, increased sequencing length, and decreased GC-bias. EM-seq enables the study of the previously unknown cannabis methylome and opens up the possibility to discover critical and exciting regulatory functions for the hermaphroditic process.

  6. Discovering the Maltose Binding Protein epitope tag for Escherichia coli expressed proteins (2018)

    Maltose Binding Protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The anti-MBP monoclonal antibody B48 binds tightly and has no crossreactivity to other proteins in an E. coli lysate. For all these criteria, the MBP tag provides a useful epitope for fusion proteins expressed in E. coli.

    The co-crystal structure of MBP bound to its antibody was solved and four amino acids of MBP were found to define the binding interaction. This epitope is the turn of an alpha helix packed by two beta strands. The failure to find a linear epitope by phage display suggests the helix-turnsheet is important in defining the smallest MBP epitope. Fusion of various fragments of MBP to the glutathione S-transferase protein was engineered in order to identify the smallest fragment, still recognized by the anti-MBP antibody. Further engineering of the epitope to stabilize and minimize the tag is in progress.

  7. The Effect of Base Modification on RNA Polymerase and Reverse Transcriptase Fidelity (2018)

    Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications. Post-transcriptional mRNA modifications can alter gene expression or mRNA stability, and can be conserved, regulated, and implicated in various cellular, developmental and disease processes. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity, and hence, RNA sequencing data. Here, we describe the fidelity of RNA polymerization and reverse transcription of modified ribonucleotides using a fidelity assay based on Pacific Biosciences®' Single-Molecule Real-Time (SMRT®) sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine (Ψ)) were examined.

  8. Complete circular genome sequence of the Wolbachia wAlbB endosymbiont of Aedes albopictus (2018)

    Wolbachia are α-proteobacteria belonging to the order Rickettsiales. It is a maternally transmitted, intracellular symbiont of arthropods and nematodes and estimated to infect 40-60% of arthropod species. The tiger mosquito Aedes albopictus is naturally infected with Wolbachia strains wAlbA and wAlbB. Cell line Aa23 established from Aedes albopictus embryos retains only wAlbB and is used as a key model to study host-endosymbiont interactions. The available wAlbB genome with 156 scaffolds is incomplete, hampering a comprehensive analysis of the genome. We have assembled the complete circular genome of a wAlbB strain from the Aa23 cell line, from long-read PacBio sequencing data at 450X coverage. The assembled circular chromosome is 1,484,007 bp in size, an increase of 321 kb over the published wAlbB genome, making it the largest sequenced Wolbachia genome to date. The annotation of the genome identified 1,207 protein coding genes, 34 tRNA, 3 rRNA and 1 tmRNA loci. The long reads enabled sequencing over complex repeat regions which have been be difficult to resolve with short-read sequencing. The availability of a complete circular genome from wAlbB will enable further biochemical, molecular and genetic analyses on this strain and related Wolbachia.

  9. Selection for proteins that overcome heatinduced lethality of ΔdegP strain (2018)

    The periplasmic protease/chaperone DegP (HtrA) plays a key role in the quality control of many proteins in the periplasm of E. coli. Proteins that fail to fold in the periplasm can be proteolysed, while others are chaperoned to their native folded state by DegP. In a ΔdegP strain, E. coli is unable to survive the protein folding stress at elevated temperatures. Utilizing this phenotype, we developed a plasmid-based selection of suppression of heat-induced lethality in a ΔdegP strain. Plasmid libraries of various prokaryotic genomes were screened for proteins that overcame heat-induced lethality. Initial hits indicate novel mechanisms of overcoming periplasmic stress, such as the periplasmic expression of a cytoplasmic GrpE homolog and the cytoplasmic expression of an unknown protein.

  10. Prediction of Golden Gate Assembly GGA Using a Comprehensive Analysis of T4 DNA Ligase End-Joining Fidelity and Bias (2018)

    The one-pot assembly of long DNA sequences from multiple component parts is key to the rapid generation of constructs for modern synthetic biology. Methods for the one-pot assembly of multiple fragments linked by short overhangs (e.g. Golden Gate) depend on accurate and unbiased ligation. Design of junctions to date largely depends on the use of rules of thumb and empirical success, rather than detailed data on ligase fidelity and bias. In this study, we have applied Pacific Biosciences Single-Molecule Real- Time sequencing technology to directly measure of the ligation frequency of every possible 5′-four-base overhang pairing in a single experiment. This comprehensive data set has been applied to predict the accuracy of Golden Gate assembly (GGA) using the Type IIS restriction enzyme BsaI. Ten fragment assemblies were designed based on the ligation data with junctions predicted to result in high or low fidelity assembly. Experimental results confirmed not only the overall accuracy, but the specific mismatch ligation errors observed and their relative frequency. The data was further used to design 12- or 24- fragment assemblies of the lac operon, which were shown to assemble with high fidelity and efficiency. Thus, ligase fidelity data allows the prediction of high-accuracy overhang pair sets with greater flexibility in design than the rules of thumb, allowing assembly of >20 fragments at high-accuracy junction points even within defined coding regions without modification of the native DNA sequence.

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