Impact of cobas PCR Media freezing on SARS-CoV-2 viral RNA integrity and whole genome sequencing analyses

Abstract

SARS-CoV-2 whole genome sequencing is a molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swabs and shipment to reference laboratories is usually required for sequencing. Cobas PCR Media transport medium facilitates high throughput SARS-CoV-2 RT-PCR analyses on cobas platforms. The manufacturer doesn't recommend freezing this transport medium because of risks of degrading molecular templates and impairing test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Sequencing performance, quality and results did not significantly differ between fresh and frozen samples (n = 10). Freezing of cobas PCR Media does not negatively affect SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories.

Keywords: Cobas PCR Media; Molecular biology; SARS-CoV-2; Transport Medium; Whole Genome Sequencing.

Fig. 1

ORF1 a/b Ct value correlation with 20X genomic sequencing coverage. Coverage of genome at 20X in relation to pre-freezing Ct value for ORF1 a/b. Less sequencing data was generated with samples with higher cycle thresholds (Ct) but freezing did not negatively impact sequencing yields.

Fig. 2

Simulated SARS-CoV-2 outbreak investigation. A simulated outbreak investigations with a back catalog of 50 SARS-CoV-2 genomic sequences from our institution (unpublished data). Phylogenetic trees were constructed using UGENE (v37) with the PHYLIP Neighbor Joining method without bootstrapping. Pre- (left) and post-freezing (right) genomic sequences show identical potential outbreak clusters within our samples.

Fig. 3

SARS-CoV-2 study isolates placement within global reference sequences. Radial rooted phylogenetic representation of SARS-CoV-2 reference sequence submitted to Nextstrain (https://nextstrain.org/sars-cov-2/). Pre- (left) and post-freezing (right) genomic sequences (red) showing identical phylogenetic placement within global reference sequences (grey).