PMC:7574920 / 20488-25754
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7574920","sourcedb":"PMC","sourceid":"7574920","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7574920","text":"Multiplexed sequencing of RT-LAMP reaction products\nOur results indicated that the colorimetric RT-LAMP assay enabled robust identification of positive samples after a 25- to 30-min incubation at 65°C. Validation of positive results, however, required confirmation that the RT-LAMP reaction led to the amplification of viral sequences. To analyze the sequences of many RT-LAMP reaction products, we established multiplexed sequencing of RT-LAMP products (LAMP-sequencing). LAMP-sequencing is based on Tn5 transposase tagmentation (17) and sample barcoding. Tagmentation enables fragmentation and direct adapter ligation of DNA samples for analysis by next-generation sequencing. We used a set of 96 barcoded adapters for tagmentation to barcode the RT-LAMP reaction products in each 96-well plate. After tagmentation, all barcoded fragments from each plate were pooled and size-selected by bead purification to remove excess adapters. A second set of barcoded primers, one per plate-pool, was then used to amplify the tagmented RT-LAMP fragments. Last, all amplified pools were combined for analysis using one next-generation sequencing run where the origin of each DNA fragment was specified by the two barcodes (Fig. 4A).\nFig. 4 Multiplexed sequencing of RT-LAMP reaction products (LAMP-sequencing).\n(A) Workflow for LAMP-sequencing is shown. A plate of 96 barcoded (BC) adapters with unique molecular identifiers (UMIs) and mosaic ends (ME) was used as a seed plate for Tn5 tagmentation of all RT-LAMP reaction products. After tagmentation, each plate was pooled individually, followed by removal of excess adapters using size selection. Each pool of tagmentation products was then amplified using primers with plate-specific barcodes, and the PCR products were analyzed by Illumina sequencing. (B) Comparison of the outcome of the three assays: LAMP-sequencing (purple, negative; green, positive; gray, too few UMIs), RT-LAMP (after 30-min incubation, y axis), and RT-qPCR (x axis). Each dot represents one sample. If a substantial number of the sequencing reads contained SARS-CoV-2 RNA, the sample was called positive (green), if not, then it was called negative (purple). For some samples (gray), no LAMP-sequencing call could be made due to too few UMIs. (See also Table 2). (C) Although the RT-LAMP assay was scored after a 30-min incubation at 65°C (left), LAMP-sequencing was performed only after the samples had been incubated for another 10 min (15 min for one plate). This panel shows the RT-LAMP assay outcome (y axis) scored after the full incubation time, whereas the RT-qPCR CT values (x axis) and LAMP-sequencing results are the same as in (B). Of the LAMP-sequencing reads obtained, 98% mapped either to the part of the viral genome targeted by the RT-LAMP primers (80.6%) or contained short k-mers derived from primer sequences (17.4%) (fig. S4). This indicated that LAMP-sequencing amplified the targeted sequences. Reads containing only primer sequences were likely to be the result of spurious amplification products as these were also formed in the absence of input RNA (Fig. 1). For quantification of individual LAMP reactions, we classified reads according to whether or not they contained viral sequences, which were not directly covered by the primers (orange segments in fig. S4A), and counted the reads for each sample (as specified by its barcode combination) (fig. S4B). For 754 of the 768 samples, we obtained enough reads to make a call (fig. S5). For the 754 samples that underwent successful LAMP-sequencing, the results confirmed all samples that scored positive on the RT-LAMP assay with a CT \u003c 30 (Fig. 4B and Table 2). For the two samples with a negative RT-qPCR result that scored positive on the RT-LAMP assay (Fig. 3), the LAMP-sequencing call agreed with the RT-qPCR result and thus corrected the RT-LAMP result.\nTable 2 Summary of LAMP-sequencing results.\nThe cross tabulation of RT-qPCR and RT-LAMP assay results shown in Table 1 have been split into samples where sequencing of RT-LAMP reaction products (LAMP-sequencing) was positive (Pos), negative (Neg), or inconclusive (too few reads) (see also Fig. 4).\nRT-LAMP\nCT Pos Neg Sum\nLAMP- sequencing Pos RT-qPCR Pos 0–25 49 0 49\n25–30 28 0 28\n30–35 4 0 4\n35–40 0 0 0\nNeg Neg 0 0 0\nNeg RT-qPCR Pos 0–25 0 0 0\n25–30 0 2 2\n30–35 0 16 16\n35–40 0 16 16\nNeg Neg 2 637 639\nToo few reads RT-qPCR Pos 0–25 2 0 2\n25–30 0 0 0\n30–35 0 0 0\n35–40 0 0 0\nNeg Neg 0 12 12\nSum 85 683 768 LAMP-sequencing was performed using the RT-LAMP samples after a prolonged incubation of 40 min at 65°C. At this time point, many of the negative samples and also samples with a CT between 30 and 40 had turned yellow. LAMP-sequencing eliminated all of these samples (Fig. 4C). This indicated that even for the RT-qPCR–positive samples with a CT between 30 and 35, the color change that took place at time points \u003e 30 min was caused by spurious amplification products and not by late amplification of viral sequences. These results therefore confirmed that LAMP-sequencing was able to assess the results of multiple RT-LAMP reactions in parallel and to identify false-positive samples in the colorimetric RT-LAMP 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