Index Adaptor and Index Primer Pooling Guidelines (NEB #E9500 and #E9530)

To download a sample sheet with the index sequences, refer to the Usage Guidelines located in the “Protocols, Manuals & Usage” tab on the product page for the NEBNext Direct Genotyping Solution Target Enrichment Kit (NEB #E9530),

Tables 3.1 and 3.2 list valid barcode combinations that can be sequenced together based on color balance guidelines for lllumina sequencers. Low-complexity barcode combinations that do not maintain the recommended color balance could lead to failed sequencing of the sample indexes.

Table 3.1 lists some valid indexed 5 ́ adaptor combinations (up to 8-plex) that can be sequenced together for the inline sample index. For combinations > 8, choose any column and add any additional combinations as needed.

Table 3.2 lists some valid indexed primer combinations (up to 4-plex) that can be sequenced together for the pool index in the Illumina i7 site. For combinations > 4 choose any 4-plex combination and add any other index as needed.

Table 3.1: Valid index 5 ́ adaptor combinations for NEB #E9500B-S.




Not recommended


A6, B6, C6, and D6
A12, B12, C12, and D12
B6, C6, D6, and E6
B12, C12, D12, and E12
C1, D1, E1, and F1
C7, D7, E7, and F7
E4, F4, G4, and H4
E10, F10, G10, H10


A1, B1, C1, D1, E1
A6, B6, C6, D6, E6
A7, B7, C7, D7, E7
A12, B12, C12, D12, E12
B1, C1, D1, E1, F1
B6, C6, D6, E6, F6
B7, C7, D7, E7, F7
B12, C12, D12, E12, F12
C1, D1, E1, F1, G1
C2, D2, E2, F2, G2
C4, D4, E4, F4, G4
C7, D7, E7, F7, G7
C8, D8, E8, F8, G8
C10, D10, E10, F10, G10
D4, E4, F4, G4, H4
D10, E10, F10, G10, H10



Any 5 plex plus 1-2 adjacent wells from the same column


Any column

Table 3.2: Valid index primer combinations for NEB #E9530B-S.




DGS01 and DGS02
DGS03 and DGS04
DGS05 and DGS06
DGS07 and DGS08


DGS01, DGS02 and DGS03
DGS02, DGS03 and DGS04
DGS03, DGS04 and DGS05
DGS04, DGS05 and DGS06
DGS05, DGS06 and DGS07
DGS06, DGS07 and DGS08


DGS01, DGS02, DGS03 and DGS04
DGS01, DGS04, DGS05 and DGS08
DGS02, DGS03, DGS04 and DGS05
DGS02, DGS04, DGS06 and DGS07
DGS03, DGS04, DGS05 and DGS06
DGS03, DGS05, DGS07 and DGS08
DGS04, DGS05, DGS06 and DGS07
DGS04, DGS05, DGS07 and DGS08
DGS05, DGS06, DGS07 and DGS08