Table 1: Total RNA for NGS library preparation has been extracted using Siemens VERSANT kPCR from 10 nasal swabs ( One sample that tested negative for SARS-Cov-2 used as negative control) and two SARS CoV-2 virus isolates as positive controls. Libraries were prepared using Trio RNA-Seq and sequenced on Illumina Novaseq 2x150bp. The sequencing results demostrate high sequence coverage for SARS-CoV-2 from ultra low input samples, indicating high sensitivity of the Trio RNA-Seq workflow from low abundant viral samples. Complete viral sequence coverage can be achieved with 100M reads for Ct<27, 50M reads for Ct<25 and 10M reads for Ct<22. The final library yield is consistent (with an average of 28 nM) across the different input amounts showing the robustness of the SPIA amplification combined with the library preparation, making it suitable for processing samples of widely varying inputs and viral load.
Graph 1: The average read distribution across the nasal swab samples shows that there is high bacterial background along with host (human) seqeunces. The Trio RNA-Seq library prep comes integrated with uninformative transcript depletion to enable enrichment of viral sequence of interest. Data have been normalized removing the unaligned reads.