Barcode adapter design. Validated barcode sequences are added to the end of the 454 A adapter via DNA synthesis (Integrated DNA Technology). The lengths of each portion of the adapter and the approximate length of the insert are indicated. Validated barcodes are exactly 11 flows in length and range from 5 to 8 bases. emPCR, emulsion PCR.

Robust, optimized plate-based acoustic shearing of genomic DNA. (a) Effect of time on shearing profile. Agilent Bioanalyzer traces of 3 μg human genomic DNA (Promega) diluted in 100 μl, aliquoted into an ABI PRISM™ Optical Reaction plate and sheared in the Covaris™ E210 under standard plate conditions (duty cycle = 5, intensity = 5, cycles per burst = 500) for increasing amounts of time (n = 3 for each timepoint). (b) Incomplete shears recovered by re-shearing. (i) Average shearing distribution (n = 27) of samples sheared for 100 seconds under standard conditions. (ii) An example of incomplete shearing seen in three attempts under standard conditions. (iii) Resultant fragment pattern after reshearing from (ii) with standard conditions. Each shear profile signal is plotted normalized to the maximum ladder fluorescence for the Bioanalyzer chip upon which the sample was run. (c) Dual high and low cutoff size-selection using para-magnetic beads (SPRI). Human genomic DNA (3 μg) was sheared under standard conditions, producing fragments ranging in size from less than 100 bp to approximately 4 kb (i). This shear product then underwent a 0.5× Solid Phase Reversible Immobilization (SPRI) reaction in which high molecular weight fragments were preferentially bound (ii). The supernatant was removed to a second tube and underwent a second 0.7× SPRI reaction where fragments below 300 bp were removed in the supernatant (iii). Fragments in the desired size range of 300 to 1,000 bp were eluted from the beads (iv).