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Recently I created a primer design for immunoglobulin heavy chain rearrangement using Illumina TruSeq LT adapters and indexes. I'm using PCR primers from a multiplexed Sanger sequencing assay that my company currently uses and it works.
I'm somewhat uncertain where to attach the regions of interest to the primers as I plan on doing a single PCR. The typical designs for the adapters consist of a forked adapter however I didn't want to do separate hybridization and adapter ligation steps.
In my first design which amplified in a KAPA qPCR library quant but failed to generate clusters, I placed the Universal adapter upstream from the forward PCR primer sequence. The reverse-complement of the indexed adapter is also upstream of the reverse PCR primer sequence.
The adapter sequences I used were as follows:
Universal adapter (P5):
5-aat gat acg gcg acc acc gag atc tac act ctt tcc cta cac gac FORWARD-PCR-PRIMER-3
Indexed adapter (P7):
5-caa gca gaa gac ggc ata cga gat NNNNNN gtg act gga gtt cag acg tgt REVERSE-PCR-PRIMER-3
Is there something I'm missing here?
I'm somewhat uncertain where to attach the regions of interest to the primers as I plan on doing a single PCR. The typical designs for the adapters consist of a forked adapter however I didn't want to do separate hybridization and adapter ligation steps.
In my first design which amplified in a KAPA qPCR library quant but failed to generate clusters, I placed the Universal adapter upstream from the forward PCR primer sequence. The reverse-complement of the indexed adapter is also upstream of the reverse PCR primer sequence.
The adapter sequences I used were as follows:
Universal adapter (P5):
5-aat gat acg gcg acc acc gag atc tac act ctt tcc cta cac gac FORWARD-PCR-PRIMER-3
Indexed adapter (P7):
5-caa gca gaa gac ggc ata cga gat NNNNNN gtg act gga gtt cag acg tgt REVERSE-PCR-PRIMER-3
Is there something I'm missing here?