An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples.

Infectious bronchitis virus (IBV) causes a highly contagious respiratory disease in chickens, leading to significant economic losses in the poultry industry worldwide. IBV exhibits a high mutation rate, resulting in the continuous emergence of new variants and strains. A complete genome analysis of IBV is crucial for understanding its characteristics. However, it is challenging to obtain whole-genome sequences from IBV-infected clinical samples due to the low abundance of IBV relative to the host genome. Here, we present a novel approach employing next-generation sequencing (NGS) to directly sequence the complete genome of IBV. Through in silico analysis, six primer pairs were designed to match various genotypes, including the GI-19 lineage of IBV. The primer sets successfully amplified six overlapping fragments by long-range PCR and the size of the amplicons ranged from 3.7 to 6.4 kb, resulting in full coverage of the IBV genome. Furthermore, utilizing Illumina sequencing, we obtained the complete genome sequences of two strains belonging to the GI-19 lineage (QX genotype) from clinical samples, with 100% coverage rates, over 1000 × mean depth coverage, and a high percentage of mapped reads to the reference genomes (96.63% and 97.66%). The reported method significantly improves the whole-genome sequencing of IBVs from clinical samples; thus, it can improve understanding of the epidemiology and evolution of IBVs.

Improved genomic resources for the black tiger prawn (Penaeus monodon).

World production of farmed crustaceans was 7.8 million tons in 2016. While only making up approximately 10% of world aquaculture production, crustaceans are generally high-value species and can earn significant export income for producing countries. Viet Nam is a major seafood producing country earning USD 7.3 billion in 2016 in export income with shrimp as a major commodity. However, there is a general lack of genomic resources available for shrimp species, which is challenging to obtain due to the need to deal with large repetitive genomes, which characterize many decapod crustaceans. The first tiger prawn (P. monodon) genome assembly was assembled in 2016 using the standard Illumina PCR-based pair-end reads and a computationally-efficient but relatively suboptimal assembler, SOAPdenovo v2. As a result, the current P. monodon draft genome is highly fragmented (> 2 million scaffolds with N50 length of <1000 bp), exhibiting only moderate genome completeness (< 35% BUSCO complete single-copy genes). We sought to improve upon the recently published P. monodon genome assembly and completeness by generating Illumina PCR-free pair-end sequencing reads to eliminate genomic gaps associated with PCR-bias and performing de novo assembly using the updated MaSurCA de novo assembler. Furthermore, we scaffolded the assembly with low coverage Nanopore long reads and several recently published deep Illumina transcriptome paired-end sequencing data, producing a final genome assembly of 1.6 Gbp (1,211,364 scaffolds; N50 length of 1982 bp) with an Arthropod BUSCO completeness of 96.8%. Compared to the previously published P. monodon genome assembly from China (NCBI Accession Code: NIUS01), this represents an almost 20% increase in the overall BUSCO genome completeness that now consists of more than 90% of Arthropod BUSCO single-copy genes. The revised P. monodon genome assembly (NCBI Accession Code: VIGR01) will be a valuable resource to support ongoing functional genomics and molecular-based breeding studies in Vietnam.