Pacbiodevnet

The complete genome sequence was determined for E. hirae R17, and published previously (Peng et al., 2016a). The genome of E. hirae R17 was sequenced using the Pacific Biosciences RS II sequencing platform (Pacific Biosciences, Menlo Park, CA, USA). Single‐molecule real‐time (SMRT^®) sequencing was conducted using the C4 sequencing chemistry and P6 polymerase with one SMRT^® cell. De novo assembly of the PacBio reads were carried out using continuous long reads (CLR) following the hierarchical genome assembly process (HGAP) workflow (PacBioDevNet; Pacific Biosciences) as available in SMRT^® Analysis v2.3 (Chin et al., 2013). The functions of predicted proteins were annotated based on homologs (using SwissProt, http://www.uniprot.org/uniprot/), and clusters of ortholog groups were determined (using COG, http://www.ncbi.nlm.nih.gov/COG/). The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was employed to identify coding sequences (CDS) based on the best‐placed reference protein set and GeneMarkS+. The complete genome sequence of E. hirae R17 was deposited in GenBank under the accession number CP015516 (chromosome) and CP015517 (plasmid pRZ1).

The Hierarchical Genome Assembly Process (HGAP) was used to assemble sequenced genomes. De novo assembly of the PacBio read sequences was carried out using continuous long reads (CLR), followed by the HGAP workflow (PacBioDevNet; Pacific Biosciences) as available in SMRT Analysis v2.3. HGAP consists of preassembly, de novo assembly with the Celera Assembler (CA), and assembly polishing with Quiver. CA software version 7.0 was utilized in the pre-assembly step, and the PacBioRs_PreAssembler with one module and a default minimum subread length of 500 bp, minimum read quality of 0.80, and minimum subread length of 7500 bp was used for error correction of the raw data generated using the PacBio RS II platform.

The genomic DNA of HA4-1 strain was purified from overnight grown liquid cultures using Blood & Cell Culture DNA Mini Kit (Qiagen) by following manufacturer’s instructions for Gram-negative bacteria. The genome sequencing was performed by combining different strategies. Firstly, a paired-end library with an insert size of 500 bp was sequenced using an Illumina HiSeq2000 using PE100 strategy. Genome assembly was performed using Velvet (1.2.07). Then, the single molecule real-time (SMRT) sequencing was performed on the PacBio RS II platform with a 20 kb library. Sequencing reads were assembled de novo by following the Hierarchical Genome Assembly Process (HGAP) workflow (PacBioDevNet; Pacific Biosciences) available in SMRT analysis version 2.3.1 (Chin et al., 2013 (link)). For gap closure and assembly correction, a BAC library containing 1536 clones was constructed with an average insert fragment of 94 kb and the clones were validated by BAC-end sequencing and PCR.

The Hierarchical Genome Assembly Process (HGAP) was used to assemble these two sequenced genomes. De novo assembly of the PacBio read sequences was carried out using continuous long reads (CLR), followed by the HGAP workflow (PacBioDevNet; Pacific Biosciences) as available in SMRT Analysis v2.1. HGAP consists of preassembly, de novo assembly with Celera Assembler (CA), and assembly polishing with Quiver. CA software version 7.0 was utilized in the pre-assembly step, and the PacBioRs_PreAssembler with one module and a default minimum subread length of 500 bp, a minimum read quality of 0.80, and a minimum subread length of 7500 bp was used to perform error correction for the raw data generated by the PacBio RS II platform. To polish the assembled sequence from HGAP, the MiSeq read sequences were mapped using BWA v0.7.5a (Li and Durbin, 2009 (link), 2010 (link)), and the SNPs and INDELs were called and corrected by SAMtools v0.1.18 (Li et al., 2009 (link)) and an in-house script. The genomes of P. capsulatum strains CBS 134186 and ATCC 48735 were deposited in Genbank under the accession numbers JPLR00000000 and JPLQ00000000.

Genomic DNA was isolated from the strains using the QIAamp DNA Minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. For T18 and T21, the chromosome and plasmids were sequenced by the PacBio RS platform (Pacific Biosciences, Menlo Park, CA) after library construction. De novo assembly of the reads was performed using continuous long reads following the Hierarchical Genome Assembly Process (HGAP) workflow (PacBio DevNet; Pacific Biosciences, Menlo Park, CA) as available in SMRT Analysis v2.3.0. For XH1568, Long-read library preparation for Nanopore sequencing was performed with a 1D sequencing kit (SQK-LSK109; Nanopore) without fragmentation. The libraries were then sequenced on a MinION device with a 1D flow cell (FlO-MIN106; Nanopore) and base called with Guppy v2.3.5 (Nanopore). The long read and short read sequence data were used in a hybrid de novo assembly using Unicycler v0.4.8 [21 (link)], then polished by Pilon v1.23 [22 (link)]. Annotation of the assemblies was performed using the NCBI PGAP annotation pipeline [23 (link)] and checked manually.