Ligation sequencing kit

The high molecular weight genomic DNA of QG10 was extracted from the 15-day-old leaf tissues following a modified CTAB method. Whole genome sequencing was done following the standard instructions of the Ligation Sequencing Kit (Nanopore, Oxford shire, UK). The quantified DNA was randomly sheared, and fragments of ∼20 kb were enriched and purified. Then, a 20-kb library was constructed and sequenced on the Nanopore PromethION platform according to the manufacturer’s protocols (Jiang et al., 2020 (link)).
De novo genome assembly of Nanopore sequence was performed as follow: The raw Nanopore reads were error-corrected and assembled using CANU (v1.7.1) (Koren et al., 2017 (link)), followed by Smartdenovo (https://github.com/ruanjue/smartdenovo) assembly, followed by three rounds of polishing with Racon (Vaser et al., 2017 (link)), followed by three rounds with Pilon v0.3.0 using the Illumina PCR-free paired-end reads (Walker et al., 2014 (link)). Genome completeness was also assessed using the algae dataset of BUSCO v2.0 (Simão et al., 2015 (link)).

Whole genomic DNA preparations were submitted to the University of Michigan sequencing core for Illumina library preparation and paired end 150 bp Illumina NovaSeq. A subset of samples was additionally sequenced using the Oxford Nanopore Minion. Nanopore libraries were prepared using the Nanopore Ligation Sequencing Kit (SQK-RBK-004) and sequenced using R9.4.1 flow cells with Guppy 3.2.10 using fast base calling mode.

We used an optimized a Phenol:Chloroform DNA isolation protocol that generates 500 ng to 3 μg large molecular weight genomic DNA from 40 mL of in vitro blood cultures. In brief, (i) short, fragmented DNA (<60kb) is removed using Small Read Eliminator Kit (Nanopore); (ii) after DNA clean-up, we prepared sequencing libraries with Ligation Sequencing Kit (Nanopore), which adds barcode to each sample. We used the Nanopore MinION Mk1C sequencer to generate long reads. We obtained 3.5 Gb (150× genome coverage) and 2.5 Gb (105×) of long-read sequencing data for Mal31 and KH004. Fifty percent of the data obtained comprise reads with length >62Kb (N50 = 62kb) with the longest read of 485 kb.
We mapped the nanopore long reads to plasmepsin gene sequences using blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Reads that cover all three plasmepsin genes and >10kb flanking regions on both sides were then extracted and compared to 3D7 reference genome to identify the repeat unit at plasmepsin genes.