I'm trying to make homemade amplicon library preps for the Illumina MiSeq, and my fusion primers always end up being around 100bp (if not more!) by the time I add all the necessary sequences (p5/p7, indices, primer landing sites, N's for diversity, etc.). This requires I buy oligos at the 250nm scale of synthesis rather than the standard 25nm scale. When I'm buying a whole plate of 96 indices, this gets very expensive very fast!
I'm thinking of an 'assembly PCR' approach in which I order four 25nm oligos. Two short ones that contain the sequence-specific sequence fused to the Illumina sequencing primer landing sites and two additional short ones that contain the p5/p7 sequences, indices, and a bit of the primer landing site. These could all be thrown into a single PCR reaction (perhaps with a bias towards the 'outer' primers containing the p5/p7 sequence).
Of course I would have to pay attention to strandedness and directionality when designing the oligos, but I wanted to see if anyone has tried this before I spend too much time thinking about it.
This idea would also have the benefit of being able to change out the sequence-specific 'inner' primer and re-use the outer primer/indices for different experiments.
I'm thinking of an 'assembly PCR' approach in which I order four 25nm oligos. Two short ones that contain the sequence-specific sequence fused to the Illumina sequencing primer landing sites and two additional short ones that contain the p5/p7 sequences, indices, and a bit of the primer landing site. These could all be thrown into a single PCR reaction (perhaps with a bias towards the 'outer' primers containing the p5/p7 sequence).
Of course I would have to pay attention to strandedness and directionality when designing the oligos, but I wanted to see if anyone has tried this before I spend too much time thinking about it.
This idea would also have the benefit of being able to change out the sequence-specific 'inner' primer and re-use the outer primer/indices for different experiments.
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