A Comparison of Bioinformatics Pipelines for Enrichment Illumina Next Generation Sequencing Systems in Detecting SARS-CoV-2 Virus Strains

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a newly emerging virus well known as the major cause of the worldwide pandemic due to Coronavirus Disease 2019 (COVID-19). Major breakthroughs in the Next Generation Sequencing (NGS) field were elucidated following the first release of a full-length SARS-CoV-2 genome on the 10 January 2020, with the hope of turning the table against the worsening pandemic situation. Previous studies in respiratory virus characterization require mapping of raw sequences to the human genome in the downstream bioinformatics pipeline as part of metagenomic principles. Illumina, as the major player in the NGS arena, took action by releasing guidelines for improved enrichment kits called the Respiratory Virus Oligo Panel (RVOP) based on a hybridization capture method capable of capturing targeted respiratory viruses, including SARS-CoV-2; therefore, allowing a direct map of raw sequences data to SARS-CoV-2 genome in downstream bioinformatics pipeline. Consequently, two bioinformatics pipelines emerged with no previous studies benchmarking the pipelines. This study focuses on gaining insight and understanding of target enrichment workflow by Illumina through the utilization of different bioinformatics pipelines named as ‘Fast Pipeline’ and ‘Normal Pipeline’ to SARS-CoV-2 strains isolated from Yogyakarta and Central Java, Indonesia. Overall, both pipelines work well in the characterization of SARS-CoV-2 samples, including in the identification of major studied nucleotide substitutions and amino acid mutations. A higher number of reads mapped to the SARS-CoV-2 genome in Fast Pipeline and merely were discovered as a contributing factor in a higher number of coverage depth and identified variations (SNPs, insertion, and deletion). Fast Pipeline ultimately works well in a situation where time is a critical factor. On the other hand, Normal Pipeline would require a longer time as it mapped reads to the human genome. Certain limitations were identified in terms of pipeline algorithm, whereas it is highly recommended in future studies to design a pipeline in an integrated framework, for instance, by using NextFlow, a workflow framework to combine all scripts into one fully integrated pipeline.

Performance of amplicon and capture based next-generation sequencing approaches for the epidemiological surveillance of Omicron SARS-CoV-2 and other variants of concern

“…A low allele frequency could cause differences in base calling due to differences in mapping and filtering. A previous publication similarly found differences in base calling when different algorithms were applied [ 39 ]. In our cohorts, 80% of the samples processed with the amplicon-based enrichment showed no discrepancies in the sequences generated with different bioinformatic pipelines, and only one discrepancy was found for the other samples.…”

Hybrid-Capture Target Enrichment in Human Pathogens: Identification, Evolution, Biosurveillance, and Genomic Epidemiology

“…Expectedly, they found that taxon-specific panels produced the best results compared to broader-range panels (e.g., respiratory viruses panel). Beyond the choice of panels, studies also investigated and verified the efficacy of HC for SARS-CoV-2 sequences across bioinformatic pipelines [ 241 ], sequencing platforms [ 242 , 243 ], and even library preparation methods [ 244 ]. Generally, molecular techniques (e.g., metagenomic sequencing, HC, and PCR) performed comparably, but in SARS-CoV-2, as the pathogen has a relatively small genome (~30 kb), enrichment of sequences can be executed using dedicated primers for the entire genome (e.g., ARCTIC 4.1).…”

Introductory Chapter: Current Landscape of Animal and Human Virus Treatments

“…The standard tool to do that is definitely the New Generation Sequencing (NGS) instrument. NGS is crucial in the discovery of new SARS-CoV-2 variants such as delta and omicron [53]. Moreover, portable NGS instrumentation such as Oxford Nanopore ® has made the integration of bioinformatics into wet laboratory pipelines more assured than ever.…”