Plant de novo genome sequencing and assembly using Oxford Nanopore Technology | Maximilian Schmidt

London Calling 2019
Maximilian Schmidt (RWTH Aachen) discussed his team's work in de novo assembly of plant genomes with nanopore technology over the past three years, and their optimisation of DNA extraction, sequencing and bioinformatics with each project.
The team's first project was the plant Solanum pennellii (wild tomato), with a genome of ~1 Gb. They generated an optimised nuclei extraction protocol, involving de-starching the plants in the dark for 2 days; the extracted DNA was then size selected via Blue Pippin. Libraries were sequenced on the MinION device, then the data error-corrected via Canu and assembled with SMART de novo, resulting in an assembly with 899 contigs, with the largest at 12.7 Mb. Next, they moved on to the Vinca minor(lesser periwinkle) genome - "we thought we'd move on to something a bit more challenging". Rich in secondary metabolites, V. minor is a plant of high pharmaceutical interest; however, the alkaloids present can inhibit extraction of DNA. Extraction of DNA for the ~750 Mb genome proved challenging: several methods were tested, eventually settling on nuclei-based extractions with sucrose. Following the same assembly method as for S. pennelli, Pilon polishing of the data was then performed, then scaffolding with the addition of Phase Genomics Hi-C data and SALSA, resulting in "almost chromosome-scale" scaffolds.
With greater than 80% of plant genomes comprised of repetitive regions, Maximilian stressed the importance of long reads in spanning repeats and improving assembly contiguity; though the team had previously enriched for fragments over 12-15 kb, this was still insufficient for some of the largest repetitive regions. They then set about improving their sample prep methods to enrich for very long fragments. By switching from gTUBE fragmentation to needle shearing and Blue Pippin High Pass Plus size selection, they increased their read N50 from 19 kb to 44 kb and improved their yield. With this optimised prep, they set about tackling the 1.9 Gb Physalis ixocarpa (tomatillo) genome: the plant is of evolutionary interest, belonging to one of very few genera that grow in both the Old and New World. The DNA extraction protocol was adapted from that of S. pennellii, a member of the same family, with some modifications - "you can't de-starch this plant for three days because it would die." Use of the updated Ligation Sequencing Kit (SQK-LSK109) and R9.4.1 Rev D Flow Cells further improved read length and yield; this was also the team's first project in which libraries were sequenced on the PromethION device, and Maximilian noted the reduction in library preps required to generate sufficient sequencing data on PromethION flow cells. Assembly was performed with wtdbg2 and SMARTdenovo, with the addition of Hi-C data increasing contig N50 from ~1 Mb to ~4 Mb. Following several cycles of polishing via pilon bowtie and pilon bwa, the assembly reached that of an "almost reference-quality genome". Next, they used this optimised workflow to assemble the Physalis alkegengi genome. Maximilian noted the significant increase to assembly quality with the introduction of the Flip-Flop basecaller, with the quality already close to that of Canu-corrected assemblies.
As there is "no genome without genes", Maximilian then discussed the direct RNA sequencing of the tomatillo to define the genes present. After optimising an RNA extraction and mRNA purification method, using "old-school" CTAB extraction to maximise yield, they performed direct RNA sequencing of the samples. In "less than a day", reads were mapped via Minimap, structural annotation performed with Stringtie and functional annotation with the Mercator4 pipeline: two thirds of the expected genes were identified in a single run. Maximilian concluded that nanopore RNA sequencing "significantly shortens gene prediction and annotation."
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