In addition, the direction of index sequencing varies for the third-party Reverse Complement Workflow and Forward Strand Workflow. This affects how sample sheets should be set up. Figure 1 illustrates the differences and similarities in index sequencing.
Figure 1 Index Sequencing Directions and Order for G4 Sequencing and Examples for Third-party Sequencing
Index Nomenclature in This Topic
Third-Party indices can have various names. In this topic, we use the following nomenclature:
• SG Index 1 = SP1-adjacent index = 3rd Party Index 2. We refer to the third-party index that is located next to SP1 as the SP1-adjacent index or 3rd Party Index 2. This corresponds to Index 1 for the Instrument.
• SG Index 2 = SP2-adjacent index = 3rd Party Index 1. We refer to the third-party index that is located next to SP2 as the SP2-adjacent index or 3rd Party Index 1. This corresponds to Index 2 for the Instrument.
Order of Index Sequencing
The comparison of the order of index sequencing is as follows:
First Index Sequenced
Second Index Sequenced
G4 index sequencing
Index 1
Index 2
Third-party Platform
Reverse Complement Workflow
SP2-adjacent Index
SP1-adjacent Index
Third-party Platform
Forward Strand Workflow
SP2-adjacent Index
SP1-adjacent Index
Direction of Index Sequencing
The comparison of the direction of index sequencing is as follows:
SG Index 1
SP1-adjacent Index
3rd Party Index 2
SG Index 2
SP2-adjacent Index
3rd Party Index 1
G4 index sequencing
Outside in, towards the insert
Outside in, towards the insert
Third-party Platform
Reverse Complement Workflow
Inside out, towards the fragment ends
Inside out, towards the fragment ends
Third-party Platform
Forward Strand Workflow
Outside in, towards the insert
Inside out, towards the fragment ends
Differences in Sample Sheet Index Entry
This affects how to demultiplex the sample, which is specified in the sample sheet. Figure 2 and Figure 3 provide examples of how index sequences relate for G4 sequencing and third-party sequencing.
Figure 3 Sample Sheet Entries for Index Sequences from Figure 2.
The table below summarizes how to set up the G4 sample sheet, compared to the entries in a sample sheet for third-party platforms.
Note
If there is an error in the index settings in the sample sheet, the samples will not demultiplex properly. To redo demultiplexing, correct the sample sheet and follow the directions in Fixing Improper Demultiplexing in the Instrument User Guide or How do I re-demultiplex my run off-instrument.
G4 index sequencing
Third-party Platform, Reverse Complement Workflow
Third-party Platform, Forward Strand Workflow
Index 1
Reverse complement of SP1-adjacent Index, second index sequenced
Sequence of SP1-adjacent Index, second index sequenced
Index 2
Reverse complement of SP2-adjacent Index, first index sequenced
Reverse complement of SP2-adjacent Index, first index sequenced