LitCovid-sentences  

PMC:7574920 / 20488-25754 JSONTXT 9 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T138 0-51 Sentence denotes Multiplexed sequencing of RT-LAMP reaction products
T139 52-201 Sentence denotes Our results indicated that the colorimetric RT-LAMP assay enabled robust identification of positive samples after a 25- to 30-min incubation at 65°C.
T140 202-335 Sentence denotes Validation of positive results, however, required confirmation that the RT-LAMP reaction led to the amplification of viral sequences.
T141 336-472 Sentence denotes To analyze the sequences of many RT-LAMP reaction products, we established multiplexed sequencing of RT-LAMP products (LAMP-sequencing).
T142 473-556 Sentence denotes LAMP-sequencing is based on Tn5 transposase tagmentation (17) and sample barcoding.
T143 557-678 Sentence denotes Tagmentation enables fragmentation and direct adapter ligation of DNA samples for analysis by next-generation sequencing.
T144 679-797 Sentence denotes We used a set of 96 barcoded adapters for tagmentation to barcode the RT-LAMP reaction products in each 96-well plate.
T145 798-934 Sentence denotes After tagmentation, all barcoded fragments from each plate were pooled and size-selected by bead purification to remove excess adapters.
T146 935-1046 Sentence denotes A second set of barcoded primers, one per plate-pool, was then used to amplify the tagmented RT-LAMP fragments.
T147 1047-1223 Sentence denotes 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).
T148 1224-1302 Sentence denotes Fig. 4 Multiplexed sequencing of RT-LAMP reaction products (LAMP-sequencing).
T149 1303-1345 Sentence denotes (A) Workflow for LAMP-sequencing is shown.
T150 1346-1524 Sentence denotes 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.
T151 1525-1641 Sentence denotes After tagmentation, each plate was pooled individually, followed by removal of excess adapters using size selection.
T152 1642-1987 Sentence denotes 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).
T153 1988-2019 Sentence denotes Each dot represents one sample.
T154 2020-2179 Sentence denotes 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).
T155 2180-2482 Sentence denotes 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).
T156 2483-2664 Sentence denotes 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).
T157 2665-2868 Sentence denotes 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).
T158 2869-2938 Sentence denotes This indicated that LAMP-sequencing amplified the targeted sequences.
T159 2939-3105 Sentence denotes 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).
T160 3106-3404 Sentence denotes 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).
T161 3405-3483 Sentence denotes For 754 of the 768 samples, we obtained enough reads to make a call (fig. S5).
T162 3484-3660 Sentence denotes 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 < 30 (Fig. 4B and Table 2).
T163 3661-3858 Sentence denotes 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.
T164 3859-3903 Sentence denotes Table 2 Summary of LAMP-sequencing results.
T165 3904-4158 Sentence denotes The 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).
T166 4159-4166 Sentence denotes RT-LAMP
T167 4167-4186 Sentence denotes CT Pos Neg Sum
T168 4187-4244 Sentence denotes LAMP- sequencing Pos RT-qPCR Pos 0–25 49 0 49
T169 4245-4261 Sentence denotes 25–30 28 0 28
T170 4262-4276 Sentence denotes 30–35 4 0 4
T171 4277-4291 Sentence denotes 35–40 0 0 0
T172 4292-4310 Sentence denotes Neg Neg 0 0 0
T173 4311-4346 Sentence denotes Neg RT-qPCR Pos 0–25 0 0 0
T174 4347-4361 Sentence denotes 25–30 0 2 2
T175 4362-4378 Sentence denotes 30–35 0 16 16
T176 4379-4395 Sentence denotes 35–40 0 16 16
T177 4396-4418 Sentence denotes Neg Neg 2 637 639
T178 4419-4464 Sentence denotes Too few reads RT-qPCR Pos 0–25 2 0 2
T179 4465-4479 Sentence denotes 25–30 0 0 0
T180 4480-4494 Sentence denotes 30–35 0 0 0
T181 4495-4509 Sentence denotes 35–40 0 0 0
T182 4510-4530 Sentence denotes Neg Neg 0 12 12
T183 4531-4652 Sentence denotes Sum 85 683 768 LAMP-sequencing was performed using the RT-LAMP samples after a prolonged incubation of 40 min at 65°C.
T184 4653-4765 Sentence denotes At this time point, many of the negative samples and also samples with a CT between 30 and 40 had turned yellow.
T185 4766-4824 Sentence denotes LAMP-sequencing eliminated all of these samples (Fig. 4C).
T186 4825-5064 Sentence denotes 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 > 30 min was caused by spurious amplification products and not by late amplification of viral sequences.
T187 5065-5266 Sentence denotes 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 assay.