Massively Parallel DNA Sequencing Array

All possible 65,536 8-base-pair (bp) DNA sequences were assembled into a maximally compact de Bruijn sequence that was subsequently divided over 1457 oligonucleotides. Each 70-bp-long oligonucleotide contained 45 overlapping 8-mers, a 3-bp GC clamp at the 5′ end, and an identical 14-bp sequence at the 3′ end for Cy5 labeling and primer extension (Fordyce et al. 2010 (link)). Sequences were hybridized to a Cy5-labeled oligonucleotide and extended using a Klenow fragment (exo⁻) (New England Biolabs) to produce Cy5-labeled dsDNA (Fordyce et al. 2010 (link)). Cy5-labeled dsDNA was diluted to a final concentration of 1.25 μM. Each sample solution contained 0.125% of poly(ethylene glycol) (Aldrich) and D-trehalose dehydrate (Sigma) at 12.5 mg/mL in dH₂O to prevent irreversible binding of the DNA to the glass as well as for visualization during alignment of the device to the DNA array. The oligos were spotted onto epoxy-coated glass substrates (CEL Associates) with a MicroGrid 610 (Bio Robotics) microarrayer using SMT-S75 silicone pins (Parallel Synthesis). Column and row pitch corresponded to the specific device. The device that we used contained 65 columns and 64 rows with a pitch of 281.25 µm by 562.5 µm, respectively. Finally, the arrays were aligned to the microfluidic device by hand under a stereoscope and bonded overnight on a heated plate at 80°C.