I would like to sequence 384 small amplicon samples on a single MiSeq run. The amplicons are 238 bp without primers. I had an idea of using primers tagged with 10bp MIDs available from Roche, and then using the TruSeq LT kit to provide an additional level of multiplexing. The TruSeq LT kit provides unique adapter combinations for up to 24 samples, so I would need 16 unique MID-labeled primer combinations to allow multiplexing of 384 samples. So, I could then order 4 unique forward primers and 4 unique reverse primers.
The delivered reads would be demultiplexed according to the TruSeq adapters, but I would then have to demultiplex each of these "samples" further according to my own MID-labeled primers.
Since the reads would begin with the Roche MID sequences, would this introduce enough sequence complexity to prevent run failure? Also, what about including amplicons produced with regular untagged primers? These would be shorter than those amplified with the MID labeled primers, but they would be easy to identify simply due to their lack of a MID sequence. Can anyone foresee any problems with this approach? Has anyone used a similar approach to multiplex a large number of samples? Thanks.
The delivered reads would be demultiplexed according to the TruSeq adapters, but I would then have to demultiplex each of these "samples" further according to my own MID-labeled primers.
Since the reads would begin with the Roche MID sequences, would this introduce enough sequence complexity to prevent run failure? Also, what about including amplicons produced with regular untagged primers? These would be shorter than those amplified with the MID labeled primers, but they would be easy to identify simply due to their lack of a MID sequence. Can anyone foresee any problems with this approach? Has anyone used a similar approach to multiplex a large number of samples? Thanks.
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