Draft genome sequence of pathogen Pasteurella multocida isolated from rabbits having pneumonia in India

TL;DR

This paper presents the genome sequence of a Pasteurella multocida strain causing pneumonia in rabbits in India.

Contribution

The novel contribution is the complete genome sequence of PMMR212, a pathogenic strain isolated from rabbits in India.

Findings

The genome contains approximately 2.35 Mb and includes 1,288 genes.

The strain was isolated from rabbits with pneumonia in Rohtak, Haryana, India.

Abstract

In this paper, we announce the complete genome of Pasteurella multocida (PMMR212) isolated from rabbits suffering from pneumonia from Rohtak, Haryana, India. The assembly contains 1,288 genes. The genome size is 2,354,118 bp (approx. 2.35 Mb).

Figures

KMT band at 460bp.

Tables

TABLE 1. The statistics of a genome assembly

Name	Statistics
Total_length	2354118
Number of sequences	1
Longest scaffold	2354118
Shortest scaffold	2354118
N50	2354118

Full text

ANNOUNCEMENT

PMMR212 was isolated from rabbits suffering from a pneumonia outbreak in Central Animal House, Maharshi Dayanand University, Rohtak, Haryana, India (GPS: 28.876° N, 76.615° E), in August and September 2023. Pasteurella multocida was isolated from the affected lungs of rabbits on brain heart infusion (BHI) agar plate at 37°C overnight. The bacteria were cultured in BHI broth for DNA isolation at 37°C overnight. DNA isolation was performed using HIMEDIA HiPurA Bacterial Genomic DNA Purification Kit according to the manufacturer’s instructions and stored at −20°C for further use. The isolated DNA was confirmed using P. multocida-specific primers (KMT: KMT1SP65′-GCTGTAAACGAACTCGCCAC-3′, KMT1T75′-ATCCGCTATTTACCCAGTGG-3′) and as P. multocida (1). Figure 1 shows KMT band 460 bp that confirms presence of P. multocida.

KMT band at 460bp.

The quality of genomic DNA and double-stranded cDNA was confirmed using Qubit dsDNA estimation and agarose gel. 500 ng gDNA and 200 ng cDNA were taken forward for each sample. Library preparation was done on all samples with NEB Next Ultra II DNA library preparation kit for Illumina; Cat no: E7770 (New England Biolabs), following the manufacturer’s recommended protocol, and the sequencing was done on the S4 flow cell of NovaSeq 6000. Commercial service providers carried out library preparation and sequencing (NxGenBio Life Sciences, New Delhi, India).

DNA was delivered with ~300–400 bp insert size for every sample. The gDNA was fragmented into smaller lengths, after which it was end-repaired and A-tailed, and adapters were ligated. The products were purified and enriched through PCR to generate the final gDNA library. Library quantification was done using a Qubit Fluorometer (Invitrogen, Life Technologies, Grand Island, NY, USA). Library fragment distribution was confirmed on the HSDNA kit using TapeStation (Agilent Technologies, USA). The tagged gDNA libraries were pooled at equivalent ratios and loaded onto the c-bot automated system for cluster generation. After cluster generation, the libraries were loaded onto an Illumina S4 Flow Cell of the Illumina NovaSeq 6000 Sequencing system, and sequencing was performed using 2 × 151 bp paired-end chemistry. Following sequencing, the samples were demultiplexed, and the indexed adapter sequences were removed using the CASAVA v1.8.2 software, Illumina Inc (2). .

After adapter trimming, the Unicycler (v 0.5.1) assembles filtered reads for a de novo assembly. RagTag (v2.1.0) was used to scaffold de novo assembly using the P. multocida strain 10,2426 reference genome. RagTag does homology-based mis-assembly correction, homology-based assembly scaffolding, patching, and scaffold merging (3) (Table 1).