Pacbio rsii sequencing platform

P. multicystogenum AS2 (Kawakami and Hagiwara, 2008 (link)) was grown as described above, but after growth cells were plated on non-nutrient agar and additionally starved overnight at 4 °C and 2 h at 21 °C to clear remaining bacteria. Genomic DNA was isolated from purified nuclei as described previously (Gloeckner et al., 2016 (link)). A PacBio 20/30 kilobase (kb) genomic DNA library was prepared from P. multicystogenum genomic DNA and sequenced using the PacBio RS II sequencing platform by the Earlham Institute, Norwich, UK. Sequencing was performed with C4-P6 chemistry on 1 SMRT cell yielding 15x coverage of the 30 megabase genome. Data quality control, basecalling, and formatting as well as HGAP data quality control were performed at the Earlham Institute. Raw reads were assembled using Canu (Koren et al., 2017 (link)) with a parameter setting recommended for low coverage (<20x) data. Specifically, the corrected error rate of reads was set to be 0.075, with other parameters set as default. The final assembly consisted of 596 contigs, totalling about 30 megabases, with an N50 of 81.6 kilobases, and is available from Genbank as bioproject PRJNA495730.

The M10M26 monospore strain was sequenced using Illumina and PacBio sequencing scheme. Three insert fragments of 270 bp (PE150, 4G raw data, Illumina Hiseq 4000, Illumina Inc., San Diego, CA, USA) and one 800 bp (PE125, 4G raw data, Illumina Hiseq 4000) PCR-free library were selected for paired-end sequencing, and one 8 Kb and one 10 Kb mate-pair library were selected for jumping sequencing (MP50, 4G raw data, Illumina Hiseq 4000). The PacBio RSII sequencing platform (PacBio P6-C4, Menlo Park, CA, USA) was used for third-generation sequencing, and the raw data of two SMRT cells were generated from the length of the 20 Kb library. Meanwhile, the RNA-sequencing of the M10 strain was carried out with an insert fragment length 270 bp (PE150, 6G raw data, Illumina Hiseq 4000). All reads have been deposited in the SRA at NCBI, under accession numbers SRR10230725–SRR10230733.