Gs-flx (whole Process)

Library Prep Double-stranded sequencing templates are blunt end repaired, and two universal adapters, A and B, are ligated to their ends.

Adapter B

Adapter A 3’

5’ 3’

5’

PCR Amplification site

20bp

20bp

4bp

4bp

20bp

Template (~80bp)

20bp

TCAG Streptavidin bead, to isolate only molecules carrying Adapter A and B

GS-FLX Sequence Primer

Adapter B 3’

Adapter A

sstDNA separated by Melt method

Oligonucleotidecoated sepharose beads

5’

Emulsion Setup

Reaction vessels

A total of 50 000 beads are required for loading onto the wells of a •After amplication, the emulsion is broken while 16th 454 FLX picotitre plate region, and the sequencing reaction the PCR products remain attached to the beads. is performed by flowing nucleotides over the plate and measuring light emissions. •Since most beads remain empty in emPCR, amplied beads are isolated through a bead Beads carrying multiple amplicons produce mixed signals, which enrichment procedure are recognized and filtered out by the run-processing software.

GS-FLX Sequencing

Emulsion Breaking and Enrichment