The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing  Kuiama Lewandowski, Andrew Bell, Rory Miles, Simon Carne, David Wooldridge, Carmen Manso, Nicola Hennessy, Daniel Bailey, Steven T. Pullan, Saheer Gharbia, Richard Vipond  The Journal of Molecular Diagnostics  Volume 19, Issue 2, Pages 303-312 (March 2017) DOI: 10.1016/j.jmoldx.2016.10.005 Copyright © 2017 Terms and Conditions

Figure 1 Quantitative RT-PCR of observed genome copies recovered from manual, EZ1, MagNA Pure, easyMag, and QIAsymphony extraction platforms. Reactions were performed in duplicate on triplicate extractions from a titration series, at 106, 105, and 104 viral copies per mL, extracted with or without Triton X-100. Error bars represent means ± SD. n = 6. The Journal of Molecular Diagnostics 2017 19, 303-312DOI: (10.1016/j.jmoldx.2016.10.005) Copyright © 2017 Terms and Conditions

Figure 2 Percentage of reads mapped to the hazara virus reference genome. Reactions were performed in triplicate on extractions at 106 and 105 viral copies per mL, extracted with or without Triton X-100 using manual, EZ1, MagNA Pure, easyMag, and QIAsymphony extraction platforms. Error bars represent means ± SD. n = 3. The Journal of Molecular Diagnostics 2017 19, 303-312DOI: (10.1016/j.jmoldx.2016.10.005) Copyright © 2017 Terms and Conditions

Figure 3 Composite figure containing representative images of amplicon PCR on control and storage samples. Duplicate samples at 106 copies per mL were stored for 1 hour, 1 day, 1 week, or 1 month at room temperature, −20°C, or −80°C in buffer AVL with or without Triton X-100. Primers were designed to target the hazara virus L segment and amplify products of sizes 180 to 2874 bp (Table 2). Assay control reactions were performed on a high titer viral RNA stock. Reactions were performed in duplicate. M, marker. The Journal of Molecular Diagnostics 2017 19, 303-312DOI: (10.1016/j.jmoldx.2016.10.005) Copyright © 2017 Terms and Conditions

Supplemental Figure S1 Composite figure containing percentage of reads taxonomically assigned to microbes and host. Reactions were performed in triplicate using manual, EZ1, MagNA Pure, easyMag, and QIAsymphony extraction platforms. A: Extractions at 106 viral copies per mL, extracted without Triton X-100. B: Extractions at 105 viral copies per mL, extracted without Triton X-100. C: Extractions at 106 viral copies per mL, extracted with Triton X-100. D: Extractions at 105 viral copies per mL, extracted with Triton X-100. Experiments were performed in triplicate at each concentration ± Triton X-100 (A–D). Error bars represent means ± SD (A–D). The Journal of Molecular Diagnostics 2017 19, 303-312DOI: (10.1016/j.jmoldx.2016.10.005) Copyright © 2017 Terms and Conditions