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BackgroundNext generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process.ResultsIn this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data.ConclusionsWe successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.

Climate change necessitates the development of CO2 neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from Myxococcus xanthus and a butyryl-CoA reductase from Clostridium acetobutylicum. The best plasmid and Escherichia coli strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO2. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization.

Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into 16, 17, and 18 carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing 16 carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel 16 carbon terpenes by GC-QTOF. On the basis of the available farnesyl pyrophosphate analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.

The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

Cyanobacteria have the potential to produce bulk and fine chemicals and members belonging to Nostoc sp. have received particular attention due to their relatively fast growth rate and the relative ease with which they can be harvested. Nostoc punctiforme is an aerobic, motile, Gram-negative, filamentous cyanobacterium that has been studied intensively to enhance our understanding of microbial carbon and nitrogen fixation. The genome of the type strain N. punctiforme ATCC 29133 was sequenced in 2001 and the scientific community has used these genome data extensively since then. Advances in bioinformatics tools for sequence annotation and the importance of this organism prompted us to resequence and reanalyze its genome and to make both, the initial and improved annotation, available to the scientific community. The new draft genome has a total size of 9.1 Mbp and consists of 65 contiguous pieces of DNA with a GC content of 41.38% and 7664 protein-coding genes. Furthermore, the resequenced genome is slightly (5152 bp) larger and contains 987 more genes with functional prediction when compared to the previously published version. We deposited the annotation of both genomes in the Department of Energy's IMG database to facilitate easy genome exploration by the scientific community without the need of in-depth bioinformatics skills. We expect that an facilitated access and ability to search the N. punctiforme ATCC 29133 for genes of interest will significantly facilitate metabolic engineering and genome prospecting efforts and ultimately the synthesis of biofuels and natural products from this keystone organism and closely related cyanobacteria.

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis1. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds1. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology2,3. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs)4. However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown5,6. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including conserved scaffolding proteins unique to the Neocallimastigomycota. High-quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single-molecule technology. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure-an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.

Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism.

Generating sequence data of a defined community composed of organisms with complete reference genomes is indispensable for the benchmarking of new genome sequence analysis methods, including assembly and binning tools. Moreover the validation of new sequencing library protocols and platforms to assess critical components such as sequencing errors and biases relies on such datasets. We here report the next generation metagenomic sequence data of a defined mock community (Mock Bacteria ARchaea Community; MBARC-26), composed of 23 bacterial and 3 archaeal strains with finished genomes. These strains span 10 phyla and 14 classes, a range of GC contents, genome sizes, repeat content and encompass a diverse abundance profile. Short read Illumina and long-read PacBio SMRT sequences of this mock community are described. These data represent a valuable resource for the scientific community, enabling extensive benchmarking and comparative evaluation of bioinformatics tools without the need to simulate data. As such, these data can aid in improving our current sequence data analysis toolkit and spur interest in the development of new tools.