Multiplexing Strategy with NEBNext Oligos/Adaptors

NEBNext Multiplex Oligos Selection Chart

	Single Index	Dual Index	Unique Dual Index	Unique Dual Index UMIs
NEB Products	NEB #E7335, NEB #E7500, NEB #E7710, NEB #E7730, NEB #E6609	NEB #E7600, NEB #E7780	NEB #E6440, NEB #E6442, NEB #E6444, NEB #E6446, NEB #E7140	NEB #E7395, NEB #E7416
Contains UMI	No	No	No	Yes
Addresses index hopping	No	No	Yes	Yes
Indexing Strategy	Index Primer	Index Primer	Index Primer	Index Adaptor
Applications	DNA-seq, RNA-seq (except Small RNA)	DNA-seq, RNA-seq (except Small RNA)	DNA-seq, RNA-seq (except Small RNA)	PCR-free DNA-seq, RNA-seq (except Small RNA)
Number of indices for multiplexing	up to 96	up to 384	up to 384	up to 96
Compatible with EM-seq	Yes*	Yes*	Yes*	No
Compatible with EpiMark bisulfite sequencing	Yes**	Yes**	Yes**	No**
Number of sets available; formats and indices available	Five; Sets 1-4 (12 indices/set): Individual vials 96 Index: Premixed plate	Two; individual vials containing 8 i5 primers and 12 i7 primers for combinatorial mixing	Four; 96 index combinations, premixed in foil-sealed 96-well plates; Including a version for EM-seq (up to 120 indices, either 96-well plate or 24 vial format)	One; 96 index adaptor combinations, premixed in foil-sealed 96 well plate (DNA-seq OR RNA-seq) and primers

* Requires the use of the EM-seq Adaptor; Single, dual and unique dual index are all compatible; NEB recommends using the Unique Dual Index Primers found in the NEBNext Enzymatic Methyl-seq Kit (NEB #E7120) or the NEBNext Multiplex Oligos for EM-seq (NEB #E7140), both supplied with the NEBNext EM-seq Adaptor; For higher levels of multiplexing, Unique Dual Index Primers Sets 3 and 4  (NEB #E6444 and #E6446) are also validated for EM-seq.

**  Requires use of NEBNext EM-seq adaptor from NEBNext Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs, #E7140S/L or NEBNext methylated adaptor from NEBNext Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1, #E7535S/L) .