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A) Random primers with 5’ barcoded adapters bind to denatured DNA templates (Figure 1). Polymerase extends the primer, making a copy of the DNA template and terminating polymerization with a biotinylated ddNTP.
B) Products are captured on streptavidin coated magnetic beads and washed to remove excess reactants.
C) A second 5’ adapter tailed primer is used with a strand displacing polymerase to convert the captured template into a dual adapter library. The primer bound closest to the magnetic bead will extend and displace primers bound downstream of the bead.
D) The dual adapter library remains bound to the beads while excess reactants and displaced products are washed away.
E) Low cycle PCR is used to amplify the products and add additional barcodes. The High Throughput version labels individual samples in a 96 well plate. After the initial labelling, products from all wells are pooled prior to the streptavidin bead capture step. 96 samples are converted into a NGS library in a single tube. An optional plate barcode is added in the index read position to allow for multiple 96 sample plates to be sequenced simultaneously.Order Riptide HT-RLP
High Throughput workflow: 96-960 Samples
After the initial primer extension and termination reaction, barcoded products from all 96 samples are pooled. All downstream processing occurs in a single tube (Figure 2). Each plate of 96 samples is further barcoded (index read) during PCR to allow for multiple 96 sample plates to be sequenced simultaneously on the same flow cell. The kit contains enough reagents for 960 samples.Order Riptide HT-RLP
The first read has 20 bases of synthetic sequence consisting of an 8 base pair barcode and and a 12 base pair random sequence (Figure 3). The remaining bases of the first read are genomic template sequence. Read 2 has 8 bases of synthetic sequence consisting of 8 bases of random sequence. The remaining bases in read 2 are genomic template sequences. The index read corresponds to one of ten Illumina index barcodes. The sample demultiplex tool is recommended for read processing prior to downstream pipelines. For detailed information, please see the demultiplex analysis tool.
Order Riptide HT-RLP