The Illumina BeadArray assays use oligonucleotides conjugated to bead types to measure specific target sequences, measuring multiple beads per bead type.
Analysis software examines the raw reads using various quality filters for removing poor quality sequences and mixed sequences that contain more than one initial DNA fragment per bead.
One platform, developed by 454 Life Sciences Corporation, the 454 GS-FLX sequencer, is capable of producing 100 Mb of sequence data with an average read length of 250 bp per bead in a 7-h run [ 32].
The volume of library to add to an aliquot of Sequence Capture Beads is calculated using the equation (1): (1) μL of library = molecules per bead × 10 million beads library concentration molecules / μL The steps from emulsion formation to biotin streptavidin-enrichment were carried out according to the manufacturer's protocols.
Table 1 shows the number of cells entrapped per bead, ethanol production, productivity and Y p/x, employing 750 beads in each experiment.
This ratio (1 copy per bead) we assumed would favor getting a single DNA molecule per capture bead in the emulsified reaction, and was the standard we had previously used for shotgun sequencing large genomes.
Oxygen consumption depends on bead volume, flow rate, and the number of cells per bead volume.
The calculated dilution factor was 5.25 and sequencing was performed as a full titration run without bead enrichment, i.e. the run was performed on a 40×75 plate, divided into 8 sectors (a titration run uses 4 of these sectors with different numbers of DNA molecules per bead i.e. 1,4,16, and 64 respectively).
At these concentrations only a few hundred QDs were visible per bead (Figure S1D, E).
A titration run, based on DNA/bead ratios of 4 and 16 copies per bead (cpb) in the emulsion-based clonal amplification, revealed the optimal experimental set-up for the large sequencing run using enriched beads.
