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. 2022 May 9;11:e69949. doi: 10.7554/eLife.69949

Figure 2. Sequencing-based detection of SARS-CoV-2 in saliva samples.

(A) Saliva preparation. Crude saliva was inactivated via TCEP/EDTA addition and 95 °C incubation prior to RT-LAMP. (B) RT-LAMP followed by COV-ID PCR performed directly on saliva. Saliva with and without addition of 1,000 copies of inactivated SARS-CoV-2 templates was inactivated as described in (A), then used as template. (C) Alignment of sequenced reads against SARS-CoV-2 genome from COV-ID of inactivated saliva spiked with or without 1,280 virions SARS-CoV-2 per µL. All SARS-CoV-2 reads align exclusively to expected region of the N gene. Open reading frames of viral genome are depicted via gray boxes below alignment. Inset: scale shows reads per 1,000. Height of peak is provided on the right. (D) Scatter plot for the ratio of SARS-CoV-2 / (STATH +1) reads obtained by COV-ID (y axis) versus the number of virions per µL spiked in human saliva (x axis). The threshold was set above the highest values scored in a negative control (dashed line). Each circle represents an independent biological replicate. (E) COV-ID performed on clinical saliva samples. The scatter plot shows the SARS-CoV-2 / (STATH +1) read ratio (y axis) versus the viral load in the sample estimated by a clinically approved, qPCR-based diagnostic test. The threshold was set based on the negative controls shown in (D). Each circle represents an independent biological replicate.

Figure 2—source data 1. Uncropped blot for Figure 2B.

Figure 2.

Figure 2—figure supplement 1. Optimization of COV-ID in human saliva.

Figure 2—figure supplement 1.

(A) Saliva COV-ID sequence validation. Single saliva COV-ID reaction using N2 primers was sequenced by the Sanger method. (B) Validation of control human amplicons for reverse transcription loop-mediated isothermal amplification (RT-LAMP) on saliva. RT-LAMP of TCEP/EDTAinactivated saliva was performed with conventional RT-LAMP primer sets for ACTB and STATH in the presence or absence of RNase A. Bars show the mean. Individual biological replicates are shown by circles. (C) Characterization of COV-ID sequencing libraries. Breakdown of reads for sequence data presented in Figure 2D. Samples without added template consist of predominantly adapter dimers. (D) Validation of COV-ID LAMP barcodes. 32 potential barcodes were tested for LAMP primer sets indicated, incompatible barcodes are marked in red. (E) Validation of pooled PCR. COV-ID was performed on saliva samples using unique LAMP barcodes. The RT-LAMP reactions were then amplified either by individual PCR or by first pooling and then performing a single PCR on the pool. Individual biological replicates are shown by circles. Lines indicate the median and interquartile range.
Figure 2—figure supplement 2. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) on a SARS-CoV-2 synthetic calibration standard.

Figure 2—figure supplement 2.

(A) A synthetic calibration standard for SARS-CoV-2 N2 RNA was synthesized including a 7 nt divergent sequence (in red), maintaining all other LAMP primer binding sites and identical GC content. (B) RT-LAMP using COV-ID N2 primers was carried out on indicated amounts of synthetic calibration standard (SCS) RNA, showing rapid amplification down to picogram quantities of added template. (C) Total number of reads per barcode in COV-ID pool obtained by including (+) or omitting (−) the N2 synthetic calibration standard. (D) Spurious COV-ID signal for the N2 amplicon in negative control samples after normalization either to the STATH control (in absence of the synthetic calibration standard) or to the SCS. Bars indicate the mean + standard deviation.
Figure 2—figure supplement 3. Clinical validation of COV-ID on RNA from nasopharyngeal (NP) swabs.

Figure 2—figure supplement 3.

(A) COV-ID on RNA from 120 patient-derived NP swabs. COV-ID for SARS-CoV-2 and ACTB was performed using 10 unique LAMP barcodes. Pools of 10 reactions were PCR amplified and sequenced to a minimum depth of 1,000 reads. Scatterplot shows SARS/(SCS +1) ratio against mean N1/N2 Ct value from RT-qPCR assays. Red circles represent samples with Ct <31. Blue circles represent samples with Ct between 31 and 36. (B) SARS/(SCS +1) ratios for samples presented in (A) are shown grouped by their respective PCR pools. Circles are colored as in (A). The dashed line represents the threshold for samples scored as positive, corresponding to 10 times the highest signal observed in negative controls. (C) Receiver operator characteristic (ROC) plots for the results shown in (A). Sensitivity and specificity of COV-ID are shown by comparing diagnostic prediction of COV-ID against SARS-CoV-2 RT-qPCR. True positives were defined as those with average N1+/N2 qPCR Ct values less than either 40 (left panels), 36 (middle), or 31 (right). Diagonal black lines represent performance of a random classifier. (D) Precision-recall curves constructed with the same data and thresholds as in (C). Gray lines represent a baseline classifier where all tests return a positive result.