NGS on SOP-generated HRV-16-specific sequence from pure and mixed samples is slightly less sensitive than quantitative real-time RT-PCR (qrRT-PCR). NGS.

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NGS on SOP-generated HRV-16-specific sequence from pure and mixed samples is slightly less sensitive than quantitative real-time RT-PCR (qrRT-PCR). NGS on SOP-generated HRV-16-specific sequence from pure and mixed samples is slightly less sensitive than quantitative real-time RT-PCR (qrRT-PCR). Four independent tests were conducted to determine the sensitivity of the SOP. Test 1 detects HRV-16 sequence from dilutions of purified virus. Test 2 detects HRV-16 sequence from dilutions of genomic RNA. Test 3 detects HRV-16 sequence from dilutions of virus spiked into H1 HeLa cells. Test 4 detects HRV-16 sequence from genomic RNA dilutions spiked into total HeLa cell RNA. A ribosomal removal step was performed for tests 3 and 4 prior to the initiation of the SOP. For each sample, a fraction of the RNA used to initiate the SOP was subjected to qrRT-PCR analysis. (A to D) HRV-16-specific reads obtained by MiSeq (black solid lines) are plotted on the left y axis and the cycle threshold (Ct) values are plotted on the right y axis (red lines). Sequencing reads not mapping to the HRV-16 reference are also indicated (black dashed lines). Corresponding HRV-16 input PFU values are plotted on the x axis. (A) The limit of detection (LOD) for test 1 in this experiment is between 101 and 102 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (100 to 101 input PFU). (B) The LOD for test 2 in this experiment is between 101 and 102 input PFU. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (100 to 101 input PFU). (C) The LOD for test 3 in this experiment is between 102 and 103 input PFU. The corresponding LOD by qrRT-PCR is approximately 100-fold greater (100 to 101 input PFU). (D) The LOD for test 4 in this experiment is between 101 and 102 input PFU; however, single reads are detected down to an input of 10−1. The corresponding LOD by qrRT-PCR is approximately 10-fold greater (100 to 101 input PFU) when individual HRV-16 reads are not considered and approximately 10-fold less sensitive when individual HRV-16 reads are considered. Lindsey A. Moser et al. mSystems 2016; doi:10.1128/mSystems.00039-15