Depending on sequencer and in case of the HiSeq 4000 even depending on run type (single-end or paired-end) Illumina uses different approaches to sequence the indices. Please find detailed information here: indexed-sequencing-overview-guide-15057455-04-Illumina-pages1to8
The correct orientation of the barcode sequence fuehrer depends on the way the barcodes are added to the library. The gist of it is:
For barcodes added to Illumina libraries via a PCR step (e.g. Nextera; or also onto TruSeq stub-adapters):
- index 1 (i7) is always read as reverse complement of the sequence in TruSeq or Nexterastyle PCR oligos
- index 2 (i5) is read in direction of TruSeq or Nextera PCR oligos for Miseq, HS4000 SE (single-end), and NovaSeq
- index 2 (i5) is read as reverse complement of barcoded PCR oligos for NextSeq, iSeq, and HS4000 PE runs
For barcoded adapters added via ligation (e.g. standard Truseq style Y-adapters):
- index 1 (i7) is always read in direction(5’to 3′) of the sequence in TruSeq style oligo
- index 2 (i5) is read in direction of TruSeq or Nextera PCR oligos for Miseq, HS4000 SE (single-end), and NovaSeq
- index 2 (i5) is read as reverse complement of barcoded PCR oligos for NextSeq, iSeq, and HS4000 PE runs
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