Building experiential learning about DNA sequencing through algae bloom monitoring onboard R/V Sikuliaq
The warming temperatures of the oceans affect phytoplankton communities. The frequency of algae blooms increases and causes dramatic consequences for Alaskan fisheries and for coastal native communities. Indeed, Alaskan traditional diets include impacted shellfish that have accumulated toxins produced by algae. Unfortunately, freezing or cooking do not neutralize these toxins. Thus, the preservation of traditional living of native communities necessitates the implementation of monitoring programs for harmful algae blooms and toxic shellfish. In October 2018, we introduced 12 undergraduate students to field genomics while navigating the Alaskan seas aboard the research vessel Sikuliaq. Using MinIONs, students analyzed in real-time the microbial communities of seawater samples. The second edition of our workshop will occur during London Calling 2019. We will sail the Northwestern Pacific Ocean for 9 days from San Diego, CA to Seward, AK. This time of the year and route are well suited to investigate algae blooms. Building on the design of the experiential learning we implemented in 2018, we will analyze sea water for phytoplankton, collect chlorophyll content measurements and microscopic images. By integrating their sequencing results with oceanographic data and imagery, participants will have the opportunity to fathom the relevance of using Oxford Nanopore Technology for urgent environmental concerns.
C-to-U RNA editing signals revealed by nanopore direct RNAseq
RNA editing is a relevant epitranscriptome modification able to modify primary transcripts by insertions, deletions or base conversions. In mammals, it includes the deamination of adenosine in inosine or the conversion of cytosine (C) to uridine (U). While the former has been well characterized by Illumina RNAseq, the latter, carried out by APOBEC1 enzyme, is rare and only a few instances have been discovered to date. Indeed, the identification of C-to-U events by second generation sequencing is quite challenging because real C-to-U modifications are masked by C-to-T transitions due to sequencing errors. Using nanopore directed RNA sequencing, we explored C-to-U RNA editing signals comparing RNA molecules from a wild type macrophagic mouse cell line and the corresponding APOBEC1 knockout cell line. Our results on candidate positions reveal a strong C-to-U signal indicating that the nanopore direct RNAseq could be a valid alternative to Illumina cDNA sequencing for C-to-U RNA editing.
MinION for barcoding and metabarcoding in polar environments
DNA barcoding (DNA-based species identification), is, like other fields of biology, being rapidly transformed by next-generation sequencing. Because of their environmental focus, barcoding (taxonomic identification of an individual specimen of plant, animal, or fungus) and metabarcoding (identification of the plant, animal, and fungal taxa present in an environmental sample) are ripe for field-ready NGS technologies, including the MinION and emerging Oxford Nanopore products. #PolarPoo, an international, interdisciplinary research campaign begun on Twitter, is an ideal test and showcase for MinION sequencing for barcoding and metabarcoding. As a #PolarPoo pilot, we compared MinION and Illumina MySeq for sequencing identical fecal material from the Antarctic black-browed albatross (Thalassarche melanophris), an important climate change indicator species in the Antarctic. Here I report the results of this comparison and discuss the applications of nanopore sequencing in polar research.
Direct nanopore sequencing of ribosome protected mRNA fragments
Ribosome profiling provides positional information of ribosomes along coding mRNA sequences, thus representing a powerful tool for gene expression analysis at the protein synthesis level. The currently available technologies for ribosome profiling are time-consuming, requiring laborious sample processing and several days for the preparation of DNA-based libraries that i) cannot carry over all the sequence information contained in native RNA molecules, and ii) easily introduce amplification biases. Oxford Nanopore pioneered direct RNA sequencing with a nanopore-based long-read sequencing technology. Here, we present a new library prep strategy for nanopore sequencing of short RNA molecules. Our method enables the reading of 30 nt-long ribosome-protected fragments (RPFs) and allows for faster ribosome profiling experiments with the potential to deliver information on post-transcriptional sequence modifications.
Structural diversity in the budding yeast
Population genomics within the last few years has been driven by short-read sequencing. Pertaining to my species of interest, Saccharomyces cerevisiae, there has been a large number of strains with high coverage Illumina read datasets, having been released within the last two years, totaling approximately 2000. Although this allows us to have a good view of SNPs and INDELs, heterozygosity and the pangenome, we currently lack a comprehensive view of structural variation, including complex rearrangements, within our species of interest, or any species to date. Our project has taken advantage of Oxford Nanopore long-read sequencing to catalogue structural variations (SV) in ~100 strains representing a wide variety of clades, some with distinct phenotypes such as high suflite resistance in some European wine strains. To discover SVs we use de novo assembly of highly contiguous genomes which can be easily compared to a reference using Whole Genome Alignment. This SV catalogue, alongside detailed SNP and phenotype data, would give us the means to tease apart the phenotypic impact of SVs and SNPs, using CRIPSR technology to engineer the discovered SVs into a known genetic background.
Complete genomes of eighteen different Actinobacillus pleuropneumoniae reference serotypes assembled with Oxford Nanopore reads
Actinobacillus pleuropneumoniae is a Gram-negative Pasteurellaceae causing pig pleuropneumonia. Eighteen serovars with distinctive lipopolysaccharide and capsular compositions were described so far, yet complete genomes are only available for the reference strains of serotype 1, 4, and 5b. We aimed to complete this picture by sequencing the remaining reference strains with the MinION sequencer and llumina HiSeq platform. Genome assemblies were performed following two different strategies, i.e., Oxford Nanopore-only de novo assemblies polished with Illumina reads or hybrid assemblies combining Oxford Nanopore and Illumina reads. Both methods proved successful to obtain accurate circular genomes with comparable quality to PacBio-generated assemblies, as shown by comparison with the PacBio-sequenced genomes of serotype 1 and 4 reference strains from the NCBI database. Further comparative genome analysis allowed identifying common and distinctive traits of the different serovars and inferring their phylogenetic relatedness. These genomic data provide useful information for diagnostics, epidemiological studies and vaccine development.