Figure 2

Bisulfite libraries prepared using the Fluidigm Access Array system. (A) Low-throughput libraries prepared manually were observed to produce strong dominant bands of the expected size with minimal visible dimer product, when visualized by agarose gel. Size in base pairs is indicated to the left (B) Sequencing results for the percent global methylation of the control libraries prepared manually. (C) Preliminary results with the Fluidigm Access Array platform resulted in weakly amplifying sequencing libraries with prominent dimer products. (D) After extensive optimization to identify the critical parameters, pre-amplification under ideal conditions still gave variable library performance using the Access Array system, with minor differences in pre-amplification primer concentration or the number of cycles of pre-amplification leading to failed libraries (that is, lane 1 vs lane 2). Lane 1, 200 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 2, 50 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 3, 200 nM primer, 15 cycles pre-amplification, Roche HF buffer; lane 4, 200 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 5, 50 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 6, 200 nM primer, 20 cycles pre-amplification, Roche HF buffer.