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Old 01-18-2016, 03:51 AM   #2
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When libraries are constructed the length of the DNA fragment between the two adapter ends is randomly distributed, determined either by random shearing (i.e. Covaris) or random tagmentation (Nextera preps). There is no way of knowing with certainty how far index 1 is away from the read 1 sequencing primer. More importantly the index is almost certainly much farther away from the read 1 primer than could reasonably be sequenced in one read; the TruSeq Nano prep kit targets insert sizes of 350 or 550bp.

Using dedicated primers for the index reads, placed immediately upstream of the index sequence is the most efficient and accurate method for sequencing the indexes.
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