A Standardized Pipeline for Assembly and Annotation of African Swine Fever Virus Genome

Abstract

Obtaining a complete good-quality sequence and annotation for the long double-stranded DNA genome of the African swine fever virus (ASFV) from next-generation sequencing (NGS) technology has proven difficult, despite the increasing availability of reference genome sequences and the increasing affordability of NGS. A gap analysis conducted by the global African swine fever research alliance (GARA) partners identified that a standardized, automatic pipeline for NGS analysis was urgently needed, particularly for new outbreak strains. Whilst there are several diagnostic and research labs worldwide that collect isolates of the ASFV from outbreaks, many do not have the capability to analyze, annotate, and format NGS data from outbreaks for submission to NCBI, and some publicly available ASFV genomes have missing or incorrect annotations. We developed an automated, standardized pipeline for the analysis of NGS reads that directly provides users with assemblies and annotations formatted for their submission to NCBI. This pipeline is freely available on GitHub and has been tested through the GARA partners by examining two previously sequenced ASFV genomes; this study also aimed to assess the accuracy and limitations of two strategies present within the pipeline: reference-based (Illumina reads) and de novo assembly (Illumina and Nanopore reads) strategies.

Keywords: ASF; ASFV; African swine fever; African swine fever virus; next-generation sequencing; pipeline.

Figure 1

Workflow of the reference-based pipeline. User provided input files (grey oval), final output files (black oval), temporary files (light blue oval), software (blue box), in house developed algorithms (dark blue box), and databases (dark grey cylinders) are represented by the indicated shapes and colors. The generalized processes of read correction and filtering (orange), de novo assembly (yellow), reference prediction and mapping (light and dark green), error correction (grey), coverage and quality determination (light cyan), and genome characterization (pink) are boxed by the indicated colors.

Figure 2

Workflow of the de novo assembly pipeline. User provided input files (grey oval), final output files (black oval), temporary files (light blue oval), software (blue box), in house developed algorithms (dark blue box), and databases (dark grey cylinders) are represented by the indicated shapes and colors. The generalized processes of read correction and filtering (orange), de novo assembly (yellow), contig extension (light green), error correction (grey), coverage and quality determination (light cyan), and genome characterization (pink) are boxed by the indicated colors.

Figure 3

Coverage (top row) and mapping quality (bottom row) of trimmed Illumina reads that have (blue line) or have not (orange line) been processed to eliminate host reads as described in the Materials and Methods section.