I have pooled libraries before and after SureSelect targeted enrichment. The barcodes have come out fairly even (within the limitations of the very low amounts of starting material). I am now turning to having my adaptors still have the barcodes as the first six bases of the reads, but with the 'Y' adaptors used by more up-to-date illumina protocols. This has required a redesign of the adaptors. A single example with the barcode AACCTT is below:
+adaptor 1 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCT*T 3'
-adaptor 1 5' [Phos]-AGGTTAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 3'
In this case, both read 1 and read 2 will have the barcode sequence, so you need to either balance your barcodes so that the 'A', 'C', 'G' and 'T' are fairly even in the first four cycles (like in my original post with four barcodes). Alternatively, you need to change the protocol of the RTA program to use different cycles (like cycles 7-10) to set phasing and intensity values. The Illumina sales rep can show you how to do this.
+adaptor 1 5' ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCT*T 3'
-adaptor 1 5' [Phos]-AGGTTAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 3'
In this case, both read 1 and read 2 will have the barcode sequence, so you need to either balance your barcodes so that the 'A', 'C', 'G' and 'T' are fairly even in the first four cycles (like in my original post with four barcodes). Alternatively, you need to change the protocol of the RTA program to use different cycles (like cycles 7-10) to set phasing and intensity values. The Illumina sales rep can show you how to do this.
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