Tonight I watched the Oxford Nanopore Technologies (ONT) PAG Industry Workshop session titled “A single-platform solution for plant de novo genome assembly.” Jeannie Mounger, a field applications scientist with ONT, was the presenter. They noted that for plant genome assembly there are several considerations. Plant genome size varies greatly: from Mb to Gb. Polyploidy varies too, with the commercial strawberry being an octoploid! Repetitive and transposable elements in plants can be large and complicate assembly. High heterozygosity complicates assembly too. Secondary metabolites may affect genomic DNA extraction and purity. For example, polysaccharide co-precipitation. Sample complexity and quality and the diversity of samples are additional considerations. Long-read sequencing technology is beneficial for assembly. Mounger noted that methylation is ubiquitous among eukaryotes and impacts numerous biological processes. In maize, for example, CpG methylation in transcribed regions is positively correlated with transcription whereas CHG methylation is negatively correlated, explained Mounger. Transcript analysis is important for improving assemblies. Extracting from fresh or flash-frozen tissue is important for high molecular weight DNA. Mounger recommended the use of CTAB protocol based on the Carlson lysis buffer followed by QIAGEN Genomic-tip 500G. Size selection is optional and can help improve N50. The ligation Sequencing Kit was recommended for library prep. For analysis, ONT recommends filtering low quality reads with SeqKit followed by assembly with hifiasm. For degraded samples and N50s below 10 kb, they recommend using Flye for assembly. Medaka is recommended for polishing. Mounger spoke about ONT sequencing platforms available and how the PromethION flow cells provide ~30X coverage for a 3 Gb genome. Mounger also listed several resources available in the ONT community. The session ended with two examples of assemblies of tomato and maize with ONT that yielded low numbers of contigs and high quality values above 50. This twenty-minute session was packed with information, and I had to watch it twice!
