A comparison of five Illumina, Ion Torrent, and nanopore sequencing technology-based approaches for whole genome sequencing of SARS-CoV-2.

Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log10 at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Convalescent Plasma in a Patient with Protracted COVID-19 and Secondary Hypogammaglobulinemia Due to Chronic Lymphocytic Leukemia: Buying Time to Develop Immunity?

It is not exactly clear yet which type of immune response prevails to accomplish viral clearance in coronavirus disease 2019 (COVID-19). Studying a patient with chronic lymphocytic leukemia and hypogammaglobulinemia who suffered from COVID-19 provided insight in the immunological responses after treatment with COVID-19 convalescent plasma (CCP). Treatment consisted of oxygen, repeated glucocorticosteroids and multiple dosages of CCP guided by antibody levels. Retrospectively performed humoral and cellular immunity analysis made clear that not every serological test for COVID-19 is appropriate for follow-up of sufficient neutralizing antibodies after CCP. In retrospect, we think that CCP merely bought time for this patient to develop an adequate cellular immune response which led to viral clearance and ultimately clinical recovery.

Increasing diagnostic possibilities using the geneLEAD VIII platform for detection of SARS-CoV-2.

At the time SARS-CoV-2 was identified as the cause of coronavirus disease 2019 (COVID-19) no in vitro diagnostic (IVD) tests were available since it was a new virus. Very shortly after the release of the genomic sequence of SARS-CoV-2, laboratory-developed tests (LDTs) were developed, made available and implemented in several laboratories in the Netherlands and globally. In this study, the performance of an E-gene Sarbeco specific real-time reverse-transcriptase PCR (RT-PCR) was verified on the open modus of the geneLEAD VIII sample-to-answer platform. The results obtained from 134 clinical samples, of which 63 had been tested positive, showed almost complete concordance compared to the same PCR on the routine diagnostic systems and that was validated according to the national reference standard. The only discordant sample tested positive using the routine diagnostic workflow with a cycle threshold (CT) value of 37.7, while the sample tested negative using the geneLEAD VIII workflow. In addition, good performance was achieved in analyzing a blinded SARS-CoV-2 external quality assurance (EQA) panel. Implementation of the geneLEAD VIII platform as routine diagnostic tool resulted in testing 871 clinical samples with 115 positive results. In conclusion, the geneLEAD VIII SARS-CoV-2 workflow presented in this study showed excellent diagnostic performance and with a rapid turnaround time of approximately two hours it proved a valuable option for STAT SARS-CoV-2 testing in the absence of (rapid, CE-IVD) point-of-care testing platforms.