Choice of assemblers has a critical impact on de novo assembly of SARS-CoV-2 genome and characterizing variants.

BACKGROUND: Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic following its initial emergence in China. SARS-CoV-2 has a positive-sense single-stranded RNA virus genome of around 30Kb. Using next-generation sequencing technologies, a large number of SARS-CoV-2 genomes are being sequenced at an unprecedented rate and being deposited in public repositories. For the de novo assembly of the SARS-CoV-2 genomes, a myriad of assemblers is being used, although their impact on the assembly quality has not been characterized for this virus. In this study, we aim to understand the variabilities on assembly qualities due to the choice of the assemblers. RESULTS: We performed 6648 de novo assemblies of 416 SARS-CoV-2 samples using eight different assemblers with different k-mer lengths. We used Illumina paired-end sequencing reads and compared the assembly quality of those assemblers. We showed that the choice of assembler plays a significant role in reconstructing the SARS-CoV-2 genome. Two metagenomic assemblers, e.g. MEGAHIT and metaSPAdes, performed better compared with others in most of the assembly quality metrics including, recovery of a larger fraction of the genome, constructing larger contigs and higher N50, NA50 values, etc. We showed that at least 09% (259/2873) of the variants present in the assemblies between MEGAHIT and metaSPAdes are unique to one of the assembly methods. CONCLUSION: Our analyses indicate the critical role of assembly methods for assembling SARS-CoV-2 genome using short reads and their impact on variant characterization. This study could help guide future studies to determine the best-suited assembler for the de novo assembly of virus genomes.

VirusTaxo: Taxonomic classification of viruses from the genome sequence using k-mer enrichment.

Classification of viruses into their taxonomic ranks (e.g., order, family, and genus) provides a framework to organize an abundant population of viruses. Next-generation metagenomic sequencing technologies lead to a rapid increase in generating sequencing data of viruses which require bioinformatics tools to analyze the taxonomy. Many metagenomic taxonomy classifiers have been developed to study microbiomes, but it is particularly challenging to assign the taxonomy of diverse virus sequences and there is a growing need for dedicated methods to be developed that are optimized to classify virus sequences into their taxa. For taxonomic classification of viruses from metagenomic sequences, we developed VirusTaxo using diverse (e.g., 402 DNA and 280 RNA) genera of viruses. VirusTaxo has an average accuracy of 93% at genus level prediction in DNA and RNA viruses. VirusTaxo outperformed existing taxonomic classifiers of viruses where it assigned taxonomy of a larger fraction of metagenomic contigs compared to other methods. Benchmarking of VirusTaxo on a collection of SARS-CoV-2 sequencing libraries and metavirome datasets suggests that VirusTaxo can characterize virus taxonomy from highly diverse contigs and provide a reliable decision on the taxonomy of viruses.

Old tools, new applications: Use of environmental bacteriophages for typhoid surveillance and evaluating vaccine impact.

Typhoid-conjugate vaccines (TCVs) provide an opportunity to reduce the burden of typhoid fever, caused by Salmonella Typhi, in endemic areas. As policymakers design vaccination strategies, accurate and high-resolution data on disease burden is crucial. However, traditional blood culture-based surveillance is resource-extensive, prohibiting its large-scale and sustainable implementation. Salmonella Typhi is a water-borne pathogen, and here, we tested the potential of Typhi-specific bacteriophage surveillance in surface water bodies as a low-cost tool to identify where Salmonella Typhi circulates in the environment. In 2021, water samples were collected and tested for the presence of Salmonella Typhi bacteriophages at two sites in Bangladesh: urban capital city, Dhaka, and a rural district, Mirzapur. Salmonella Typhi-specific bacteriophages were detected in 66 of 211 (31%) environmental samples in Dhaka, in comparison to 3 of 92 (3%) environmental samples from Mirzapur. In the same year, 4,620 blood cultures at the two largest pediatric hospitals of Dhaka yielded 215 (5%) culture-confirmed typhoid cases, and 3,788 blood cultures in the largest hospital of Mirzapur yielded 2 (0.05%) cases. 75% (52/69) of positive phage samples were collected from sewage. All isolated phages were tested against a panel of isolates from different Salmonella Typhi genotypes circulating in Bangladesh and were found to exhibit a diverse killing spectrum, indicating that diverse bacteriophages were isolated. These results suggest an association between the presence of Typhi-specific phages in the environment and the burden of typhoid fever, and the potential of utilizing environmental phage surveillance as a low-cost tool to assist policy decisions on typhoid control.