Oyola et al. BMC Genomics 2012, 13:1 http://www.biomedcentral.com/1471-2164/13/1 To obtain a fragment-size distribution of ~100 bp to ~300 bp, the settings were: 20% duty cycle, intensity 5, and 200 cycles per burst. Illumina paired-end sequen- cing libraries were constructed using the NEBNext DNA sample preparation kit (NEB), following standard Illu- mina sample-preparation protocol with the following slight modifications. End-repair reactions (100 μl) con- tained 75 µl of sheared DNA sample, 1× T4 DNA-ligase buffer, 1 mM ATP, 0.4 mM dNTPs, 15 units of T4 DNA polymerase, 50 units of Klenow DNA polymerase, 50 units of T4 polynucleotide kinase, and were incu- bated at 20°C for 45 min. Resulting blunt phosphory- lated DNA fragments (end-repaired) were cleaned using the QIAquick PCR purification Kit following the manu- facturer’s instructions and the DNA eluted in 32 μl of buffer EB. A single A base was added (A-tailing) to the 3’ end of the end-repaired DNA fragments in a 50 μl reaction containing 1× Klenow buffer, 0.2 mM dATP, 150 units of Klenow exo- and incubated for 45 min at 37°C. A-tailed DNA fragments were cleaned using QIA- quick MiniElute PCR purification Kits following the manufacturer’s instructions and DNA eluted in 10 µl EB buffer. Pre-annealed paired-end (PE) adapter oligonu- cleotides (Illumina) were ligated to the A-tailed frag- ments in a 50 µl reaction containing 10 µl of DNA sample, 1× Quick T4 DNA-ligase buffer, 10 µl of PE- adapter mixture, 5 µl of Quick T4 DNA ligase (NEB) and incubated at 20°C for 30 min. The ligation reaction was cleaned twice with 1× Agencourt Ampure XP beads (Beckman Coulter). Aliquots were analyzed by running on an Agilent 2100 Bioanalyzer (Agilent Technologies) to determine the size distribution and to check for adap- ter contamination. Most amplified or non-amplified libraries were prepared twice (starting from same non- sheared genomic DNA) and each technical replicate sequenced independently before data analysis. All repli- cate data sets were pooled after normalizing and before analysis with the exception of coverage analysis on spe- cific genome loci (presented as box plots, Figure 4i & 4ii) where each replicate was analyzed and shown independently. Illumina PCR library amplification All PCR amplifications were performed with an MJ Research Thermo Cycler PTC-225. Illumina PE 1.0 and 2.0 primers or PE 1.0- and 2.0-derived indexing primers were used to amplify 10 ng adapter-ligated library frag- ments by PCR. From a single pool of adapter-ligated library, aliquots were amplified using standard Illumina PCR reagents or using various alternative polymerases under optimized PCR conditions. Standard Illumina PCR (50 μl) with Phusion polymerase (Thermo) contained 1× Phusion DNA polymerase master mix and 0.4 μM of each primer pair and was amplified with thermocycling Page 9 of 12 conditions of 1 min at 98°C for the initial denaturation, followed by 12 cycles of [10 s at 98°C, 30 s at 65°C, 30 s at 72°C] and a final extension for 5 min at 72°C. PCR (50 μl) with Kapa HiFi (KAPA Biosystems, South Africa) contained 1× Kapa HiFi buffer (containing TMAC) or 1× Kapa buffer B, 0.3 mM of each dNTP, 0.4 μM of each pri- mer pair, 1 unit of Kapa HiFi and was amplified with thermocycling conditions of 1 min at 98°C for the initial denaturation followed by 12 cycles of [10 s at 98°C, 1 min at 65°C] and a final extension for 5 min at 65°C. PCR (50 μl) with Kapa2G Robust (KAPA Biosystems, South Africa) contained 1× Kapa2G buffer B, 0.3 mM dNTPs mix, 0.4 μM of each primer pair, 0.4 units of Kapa2G Robust and was amplified in the presence or absence of 60 mM TMAC with the same thermocycling conditions as Phusion above. PCR (50 μl) with Platinum pfx (Invi- trogen) contained 1× Platinum pfx buffer, 0.3 mM dNTPs mix, 0.4 μM of each primer pair, 1 mM MgCl2, 0.8 units of Platinum pfx and was amplified in the pre- sence or absence of 60 mM TMAC with thermocycling conditions of 2 min at 94°C for the initial denaturation followed by 12 cycles of [15 s at 94°C, 30 s at 60°C, 30 s at 65°C] and a final extension for 5 min at 65°C. PCR (50 μl) with AccuPrime Taq HiFi (Invitrogen) contained 1× AccuPrime buffer II, 0.4 μM of each primer pair, 1 unit of AccuPrime Taq HiFi and was amplified in the presence or absence of 60 mM TMAC with the thermo- cycling conditions used for Platinum pfx. PCR products were purified with 1× Agencourt Ampure XP beads (Beckman Coulter) and eluted in EB buffer (Qiagen). PCR products were analysed by running on an Agilent 2100 Bioanalyzer (Agilent Technologies) to assess ampli- fication. Only successful amplifications were processed for sequencing. Illumina isothermal library amplification using RPA technology We used recombinase polymerase amplification (RPA) technology as provided in the TwistAmp™ DNA-amplifi- cation kit [11] to amplify 10 ng adapter-ligated library fragments. Illumina PE 1.0 and 2.0 primers or PE 1.0- and 2.0-derived indexing primers (each in 10 μM stock) were used for amplification following kit manufacturer’s instructions. Amplifications were performed twice to gen- erate technical replicates. For each sample, a rehydration solution was prepared by mixing 2.4 μl of each primer pair, 29.5 μl of rehydration buffer and 13.2 μl of template library solution. After vortexing and brief centrifugation, the 47.5 μl of rehydration solution was transferred to a freeze-dried RPA reaction complex pellet and mixed by pipetting up and down until complete resuspension was achieved. To initiate the reaction, 2.5 μl of 280 mM mag- nesium acetate was added and mixed well by vortexing fol- lowed by brief centrifugation. The reaction was allowed to