Occasionally, it may be necessary to combine libraries that have different length indexes into a single sequencing run.  Using the NEXTflex™ Barcodes, barcodes with indexes of 6-12 nucleotide length can be combined into a single run by lengthening the shorter barcodes to the length of the longest.  The chart below shows what bases to add to convert a shorter NEXTflex Barcode index into a longer NEXTflex Barcode index when using sample sheets generated with the Illumina Experiment Manager:

Barcode Additional Bases to Add to the 3’ End of Barcode
6nt barcode → 8nt barcode Add “AT”
6nt barcode → 12nt barcode Add “ATCTCG”
8nt barcode → 12nt barcode Add “ATCT”

As an example, using barcode 1 from the NEXTflex™ DNA Barcodes (cat#514101), the index sequence is CGATGT.  To use this barcode with a run containing 8nt barcodes, you would change this sequence to CGATGTAT. To use this barcode with a run containing barcodes with 12nt indexes, you would change this sequence to CGATGTATCTCG.  You can change this sequence directly in the sample sheet.

To ensure adequate diversity, libraries with different length indexes can only be combined when the barcodes with the longer indexes make up at least 50% of the reads. The Index Read quality may suffer if the majority of the indexes all end in the same few bases, so this is best done only when absolutely necessary. Also, always check for duplicates when adding bases to barcodes, as the new 6nt to 12nt index may be the same sequence as an extisting 12nt barcode. At the time of this writing, it is unknown if Illumina will troubleshoot runs that experience difficulty while employing this tactic, so users should consider this process “at their own risk.”