Plant viruses are one of the greatest problems facing crop production in the world. Its severe effects are experienced in the developing world where small-scale farming is a major source of food production and knowledge and resources for management are limited. Among other plants, cassava is one of the important crops in Africa. It is a staple food for close to one billion people in the tropics, but its production is threatened by two viral diseases, namely cassava brown streak and cassava mosaic disease. Diseased planting cuttings and whiteflies transmit these diseases. Early detection of the cassava viruses is crucial because it helps farmers make decision on which cassava variety to plant and/or where to source the clean planting materials. Our team used three portable, battery-powered devices, PDQeX DNA purification technology, developed by ZyGEM NZ Ltd, in New Zealand and MinION with MinIT sequencing platform developed by Oxford Nanopore in the United Kingdom, to produce an effective on-site diagnostic of viruses in cassava. The PDQeX system extracted a purified DNA using a cocktail of enzymes in thermo-responsive extractor cartridges inserted in a temperature control unit. Then DNA library was prepared and loaded in MinION for sequencing. MinIT did real-time base calling and identification of viruses affecting cassava field was done using a customized BLAST search.

Here we demonstrate a rapid, simplified, and portable sequencing platform using a combination of the VolTRAX V2 and the MinION. We applied this platform across two distinct research areas: 1) the environmental reservoir of antibiotic resistance; Environmental bacteria can act as reservoir of opportunistic pathogens despite a lack of exposure. Identifying specific antibiotic resistant microbes is essential for quick and appropriate treatment. 2) Detecting genomic variation in cultured cancerous cell lines. Because cancerous cell lines are often used in drug discovery and testing, it is important to understand if long-term laboratory growth might play a role in the cellular responses to various genotoxic stressors. In this proof of principle, each library was constructed by undergraduate researchers with introductory laboratory skills using the VolTRAX V2 using the guided automated library preparation. Sequencing was carried out using the MinION, the portable Oxford Nanopore DNA sequencer. We found that this combination provides a reliable and repeatable bench experience for the early career researchers

Genetically encoded reporter proteins are a cornerstone of molecular biology widely used to measure many biological activities, but the current number of unique reporters that can be used together for multiplexed tracking is small due to overlapping detection channels such as fluorescence. We therefore built an expanded library of orthogonally-barcoded nanopore-addressable protein tags engineered as reporters (NanoporeTERs), which can be read by nanopore sensors at the single-molecule level. By adapting a commercially available nanopore sensor array platform typically used for real-time DNA/RNA sequencing (Oxford Nanopore Technologies MinION), we show direct quantification of individual NanoporeTER expression levels at the protein level from engineered bacterial cultures, with little to no sample preparation. These results open new applications for multiplexed, real-time tracking of complex biological phenomena not possible with conventional protein reporters using portable, high-throughput nanopore sensor technology.

Molecular tagging is an approach to securely label physical objects of high value using DNA or other molecules. An ideal system should be inexpensive, quick and reliable to decode, and require minimal equipment. We created a novel molecular tagging system using DNA-based tags and the MinION nanopore device. In our tagging system, we first encode a 32-bit digital tag into a 90-bit codeword that is more robust to errors. We then convert it into a DNA-based molecular tag where each 1 or 0 in the codeword is represented by the presence or absence of a molecular bit (molbit). A single molbit is a DNA strand that modularly combines a unique barcode structure with a specific strand length. This allows us to classify molbits directly from the raw nanopore signal, avoiding basecalling to reduce compute time and misclassification errors. Molbits are prepared for readout at the time of tag assembly and are then stabilized by dehydration. These steps extend the shelf life of the tag, decrease decoding time, and make it robust to contamination from environmental DNA. The result is an extensible, real time, high accuracy tagging system that includes a novel approach to developing nanopore-orthogonal barcodes with applications beyond this system.

Two purposes were assessed with nanopore sequencing (i) Detection of fungal communities using 1D PCR barcoding, starting with pure cultures of Alternaria, Aspergillus, Candida, Malassezia, Microsporum canis, and Penicillum. We compared partial fungal operon (18S-ITS1-5.8S-ITS2-28S) sequencing of ~4 Kb with the whole one of ~6 Kb. Saccharomyces and Cryptococcus from the ZymoBIOMICSTM mock community were also sequenced as positive control. Amplification of partial and whole operon showed same results. (ii) Malassezia pachydermatis, commonly found in skin and may be associated to skin diseases (e.g. atopic dermatitis), was sequenced by 1D Native barcoding genomic DNA and analysed with bioinformatics tools to obtain consensus sequences and to understand if it may be similar to other Malassezia species.

Our genome expresses thousands of long noncoding RNAs (lncRNAs) that are mRNA-like RNA transcripts, which do not encode any identifiable peptide product. Although lncRNAs perform diverse roles in the cell, the overwhelming majority of them remain uncharacterized. Decoding lncRNAs function highly depends on accurate annotations, which map the precise location of lncRNA genes in the genome. Present lncRNA annotations are far from being complete and this incompleteness can have a strong impact on downstream analyses. To improve the annotation of GENCODE lncRNAs in human and mouse genomes, we developed Capture Long-read Sequencing (CLS) method that combines targeted RNA capture with long-read sequencing. Despite its benefits, CLS can produce transcripts with incomplete 5’ ends. To address this limitation, we developed Cap-CLS – an upgraded version of CLS method with the CapTrap protocol to enrich 5’ ends of transcripts. Results obtained with nanopore sequencing indicate that Cap-CLS considerably improves the annotation of 5' ends of transcripts.

The U.S. Navy operates within diverse environments that spans both maritime operating areas and shore installations including deployed ships, hospitals, overseas medical research units, and emergent field laboratories. The U.S. Naval Research Laboratory (NRL) has supported a number of research initiatives within these sites, including the employment of multi-omic approaches and analysis of the information gleaned from next-generation, high-throughput sequencing data such as genomics/metagenomics and transcriptomics to characterize discrete microorganisms or mixed microbial communities. In an effort to complement and expand current technologies and capabilities, NRL has taken advantage of the MinION’s portability and real-time data acquisition to facilitate sequencing in austere, non-traditional lab settings and/or in scenarios that require rapid responses. As an accompaniment to the MinION, NRL has also developed a portable laboratory, a kit with a small footprint that is immediately deployable and can support a complete workflow from processing of samples obtained from diverse sources, PCR amplification, library preparation, sequencing, and data analysis. This kit has been used to investigate microbial diversity onboard a naval vessel in order to characterize the communities found on the interior surfaces of an operational afloat unit with the eventual objective to track the subsequent changes to the communities of this built environment during various stages of a deployment cycle. The capability has also been demonstrated in the Navy’s expeditionary mobile laboratory, with the primary aim to augment current assays for specific pathogen detection. In addition to targeted assays, there are ongoing efforts to implement effective unbiased sequencing protocols in the field to address complex environmental and clinical samples such as water filters, biofilms, urine, and soil. Non-targeted approaches will allow for a comprehensive assessment of a samples genetic cache and enable timely taxonomic classification and identification of potential determinants associated with disease or antimicrobial resistance that impact human health.

HIV-1 and HIV-2 together with SIVs comprise the primate lentivirus family. SIVs infect many Old World African primates, both in the wild and in domesticated animals. Some SIV lineages co-evolve with their hosts, but cross-species transmission between different simian species and transmission from simians to humans can occur. Based on short sequences, SIV infection in a yellow baboon and a chacma baboon was published in the 1990s. We isolated SIV from the chacma baboon by co-culture of baboon PBMCs with human CD4+ cell lines and detecting virus by reverse transcriptase activity. High molecular weight (hmw) cellular DNA with integrated provirus was extracted in 1989, using the standard phenol-chloroform method and stored at 4°C for 20 years. Next generation sequencing was attempted, but we could not assemble a complete SIV genome. After storage for another 10 years, the archived DNA was quantified using Qubit and the DNA purity evaluated using the NanoDrop. The 1D ligation kit was used for library preparation to attempt nanopore sequencing on the GridION X5. Base called FastQ sequences, were imported in Geneious Prime 2019 and BBDuk was used for quality trimming and filtering. SIV proviral sequences were assembled to the short baboon reference sequence using the Map to Reference option of Geneious 6.0.3 Read Mapper. After using BLAST on the consensus baboon sequence, we repeated the assembly process with the most closely related SIVagm isolate. The new baboon SIV had less than 80% nucleotide sequence identity to known SIV isolates. Preliminary phylogenetic analyses indicated the baboon virus was related to the vervet subgroup of SIVagm. We are designing primers to amplify the baboon provirus to compare nanopore sequences with Sanger sequencing.

Using PromethION, we generated whole-genome long-read sequencing data of five lung cancer cell lines at the depths of 30X— in LC2/ad cells and 10X — in other cells. We identified the cancerous mutations including point mutations, large deletions and chromosomal rearrangements, which were previously reported using the short-read sequencers. In addition, we unexpectedly detected complex middle-size structural variations, which we named Cancerous Local Copy-number Lesions (CLCLs). Those CLCLs consisted of complex combinations of local inversions, duplications and micro deletions, which should invoke functional alternations of their encoding proteins. Those CLCLs occurred even in the cancer-related genes, such as STK11, NF1 and PTEN, where the short-read sequencers could not have confidently identified their aberrant structures. We further conducted the similar analyses using ten clinical lung adenocarcinoma specimens. We successfully demonstrated that those CLCLs are not specific to cultured cells, but also occurring in vivo.