Hello,
Following are the list of dual indexed illumina libraries  we have used for illumina run.But we were not able demultiplex the data by using the barcodes.Because 99% of the data going as undetermined.

Index1 ------    Index2 ( case1 )

ATGAGC  ----   TGAACCTT 

ATTCCT  ----    TGAACCTT

CAAAAG	  ----   TGAACCTT

CAACTA	  ----   TGAACCTT

CACCGG  ----   TGAACCTT

There after we tried demultiplex with  reverse compliment of index 2 and No changes in Index1(list are below ).This worked well and able to determine 98% of the data.The question is why the demultiplex only worked with reverse compliment of index2 and  not in the case1(without reverse compliment)

Index1 ------    Index2 (reverse compliment)

ATGAGC ----  AAGGTTCA

ATTCCT ----  AAGGTTCA

CAAAAG ----  AAGGTTCA

CAACTA ----  AAGGTTCA

CACCGG ----  AAGGTTCA




The index2 (i5) sequence orientation is dependent on the sequencer used. Your original i5 sequence for barcode A501 is appropriate for a HiSeq 2000/2500, MiSeq, or NovaSeq, but would need to be reverse-complemented for MiniSeq, NextSeq, or HiSeq 3000/4000 (you can see this [in the following PDF from Illumina](https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-adapter-sequences-1000000002694-06.pdf)). This turns out to be rather annoying if you run a sequencing facility with multiple types of sequencers and people provide the barcodes they use. You then either have to check the orientation manually (that's annoying) or what for failures like this and reverse-complement accordingly (this actually works OK most of the time). I ended up writing a python package for [our demultiplexing pipeline](https://github.com/maxplanck-ie/TheWhoTheWhatTheHuh) that tries to determine the proper orientation of the barcodes in each lane [by looking at what's in the BCL files produced by the sequencer](https://github.com/maxplanck-ie/TheWhoTheWhatTheHuh/blob/master/pyBarcodes.c#L18-L40).

Update: Please see the comment below from https://www.biostars.org/u/3616/ for an update on the NovaSeq!