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Multiplexing

There are many methods to segregate samples to maximize the throughput from a single sequencing run. These include separating the samples physically (loading samples in different regions of the Pico Titer Plate PTP gasket), coded separation using multiplex identifiers (MIDs) or a combination of the two. If employed, MID sequences should be used in both the A and B Fusion Primers. Using different MIDs in each of the two Fusion Primers will enable a broad range of multiplexing possibilities – up to 196–fold with 14 MIDs on each end. In all cases, bidirectional sequencing should be employed.

While other barcode sequences may be incorporated, we recommend using MIDs from the Standard 454 set in the table below (more MID sequences can be provided on request). These 10–mer sequences have been carefully engineered to avoid mis–assignment of reads and are tolerant to several errors, such as those often introduced during primer synthesis. If a greater number of MIDs are required there is an extended set of up to 156 MID sequences, please ask for more details.

These 14 MID sequences have been pre–loaded in the Amplicon Variant Analyzer Software developed by 454 to analyze data from Amplicon library sequencing.

IDMID SequenceIDMID Sequence
MID1 ACG AGT GCG T MID8 CTC GCG TGT C
MID2 ACG CTC GAC A MID9 TAG TAT CAG C
MID3 AGA CGC ACT C MID10 TCT CTA TGC G
MID4 AGC ACT GTA G MID11 TGA TAC GTC T
MID5 ATC AGA CAC G MID12 TAC TGA GCT A
MID6 ATA TCG CGA G MID13 CAT AGT AGT G
MID7 CGT GTC TCT A MID14 CGA GAG ATA C