After the index-hopping scandal (see Illumina's white paper) it sounds like the field is moving in the direction of unique dual indexing, i.e. dual indexing where every index combination is unique, so that no two libraries have the same P7 index and no two libraries have thes same P5 index. The means that instead of 12 versions of the P7 adapter and 8 versions of the P5 adapter, you'd need 96 versions of P7 and 96 versions of P5. This is an expensive investment for homebrew library protocols.
Does anyone know of experimentally validated index sequences for 96-plexity?
It would be nice to keep the 2 x 8 nt index-read lengths so we don't have to steal cycles from the actual target sequence. I see Bioo has a set of 96 barcodes that are 8 nt long, so I guess you could just reuse those same 96 on each end. But 68 out of 96 have an A or C in the first position, and a whopping 90 out of 96 have a A/C in the last position. That seems like it would be a serious problem since A/C are read by the same-color laser and Illumina says you must have color balance in your barcode combinations, even on the older machines that do four imaging cycles.
Does anyone know of experimentally validated index sequences for 96-plexity?
It would be nice to keep the 2 x 8 nt index-read lengths so we don't have to steal cycles from the actual target sequence. I see Bioo has a set of 96 barcodes that are 8 nt long, so I guess you could just reuse those same 96 on each end. But 68 out of 96 have an A or C in the first position, and a whopping 90 out of 96 have a A/C in the last position. That seems like it would be a serious problem since A/C are read by the same-color laser and Illumina says you must have color balance in your barcode combinations, even on the older machines that do four imaging cycles.
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