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Question 1: The additional sequences is needed because that is complimentary to the P5 sequence anchored to the flow cell. It is also the site for priming the Index 2 read on a MiSeq. The additional sequence is also needed for the read 1 primer in the cartridge to anneal to the correct place for the molecules. Question 2: The "GT" sequence you're referring ...


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This sounds like a job for the CombinatorialDerivation class, which is designed for representing libraries and the relationship between derived designs and the originals. About CombinatorialDerivation In SBOL 3, a CombinatorialDerivation takes a Component in its template property to represent the system that you intend to vary: Some of the Features of the ...


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You can include that info in the design, though you'd have to decide whether to include it as a number of just a factor. I've never heard of anyone including PCR cycles. I've heard of people including RIN scores, though I've never done it. If the PCA looks alright, I wouldn't worry about. I've got tons of examples of the lab people saying "I did a ...


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The order of sequencing in a dual index flow cell is: Read 1 Index 1 (I7) Index 2 (I5) Read 2 Between each step there is a switch of the primer used (Read1-primer -> Index1-primer (I7) -> Index2-primer (I5) -> Read2-primer) There are differences in how the I5 is dealt with between dual- and single-read flow cells as explained in the Illumina ...


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Module 3 does not exist during PCR amplification. Here is how NEBNext adaptor and multiplex PCR primers work. During the 1st PCR cycle, only the P7 primers are used to bind and amplify ligated DNA, generating the complementary strands that contain the binding sites for P5 primers (Universal in this case). During the second cycle of amplification, the P5 ...


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The original point of the non-complementary sequence is to allow formation of a Y-adapter after USER treatment, since that's what's needed for the Illumina chemistry to work. In reality the bottom strand may not be there in step 3, since USER has already digested one strand (creating the hairpin in step 2) and size selection has been performed. Regardless, ...


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