Draft Genome Sequence of an Isolate of Nontypeable Haemophilus influenzae from an Acute Exacerbation of Chronic Obstructive Pulmonary Disease in Tasmania.

Nontypeable Haemophilus influenzae (NTHi) is an important cause of human illness, including pneumonia and acute exacerbations of chronic obstructive pulmonary disease (COPD). We report here the draft genome of an isolate of NTHi collected from the sputum of a patient presenting with COPD in Tasmania, Australia.

characterized by a persistent reduction in lung airflow (1). It has emerged as the third leading cause of mortality, claiming more than 3 million lives worldwide in 2016 (2). In Australia, it is estimated that COPD affects 1.45 million people (3). COPD is an important disease in the state of Tasmania, where higher rates of smoking are observed with respect to the national rate (4). Nontypeable Haemophilus influenzae (NTHi) is a key pathogen that colonizes damaged airways in COPD patients and causes acute exacerbations that contribute to morbidity and mortality (5)(6)(7). Here, we present the draft assembled genome sequence of an NTHi strain isolated from a case of COPD in Tasmania.
NTHi strain RHH-38 was isolated in 2018 at the Royal Hobart Hospital in Tasmania from the sputum of a COPD patient presenting with an acute exacerbation. The sputum specimen was homogenized and cultured on chocolate blood agar plates at 35°C in a CO 2 atmosphere followed by storage at 2 to 8°C. Bacterial identification was performed using a Bruker matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer. A single colony of Haemophilus influenzae was suspended in 200 l phosphate-buffered saline (PBS), and then genomic DNA was extracted using the DNeasy blood and tissue kit (catalog number 69504; Qiagen, USA). The genomic DNA preparation was further purified using the High Pure PCR template preparation kit (catalog number 11796828001; Roche, Germany). DNA library preparation was carried out using a Nextera XT DNA library preparation kit (catalog number FC-131-1024; Illumina, USA) as described previously (8,9). Sequencing was performed using an Illumina MiSeq platform with 150-bp pairedend sequencing. In total, 1,161,034 paired-end reads were generated, representing an average read depth of 88.83-fold. Reads were trimmed of adapters using Trimmomatic (10), and de novo assembly of reads was performed with SPAdes v3.12.0 (11). All parameters were set to default except for the size of k-mers, which were manually set to 21, 33, 43, 53, 63, and 75. This resulted in the generation of a 1,914,787-bp draft genome consisting of 68 contigs (Ն200 bp) that covered 86.7% of the reference H. influenzae 86-028NP genome (12). The N 50 value was 66,703 bp, and the overall GC content was 38.1%. The genome assembly quality, including completeness with respect to the reference genome, was determined using the QUAST quality assessment tool (13).
In silico MLST analysis, performed by submission of the draft genome to the H. influenzae multilocus sequence typing (MLST) website (https://pubmlst.org/hinfluenzae/) (14), assigned RHH-38 to sequence type 422 (ST422) based on seven housekeeping genes. The draft genome was annotated using RASTtk (15)(16)(17), which identified a total of 2,019 genes consisting of 1,960 coding sequences and 59 RNA genes. Default parameters were used for all software unless otherwise specified.
In conclusion, this study presents the published genome sequence assembly of an NTHi isolate from a case of COPD in Tasmania. The application of genome sequencing has the potential to provide insights into recurrent exacerbations of COPD due to NTHi and the ability to distinguish between relapse and reinfection.
This work was conducted in accordance with ethics approval number H0016214 from the Tasmanian Health and Medical Human Research Ethics Committee.
Data availability. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number JAAECN000000000. The version described in this paper is version JAAECN010000000. The associated BioProject and BioSample accession numbers are PRJNA603840 and SAMN13942196, respectively.

ACKNOWLEDGMENT
R. KC is the recipient of a Health Tasmania Graduate Research Scholarship from the University of Tasmania.