A library of DNA fragments suitable for high-throughput sequencing with the Illumina procedure [10 (link)] was generated from 5 μl of the SGE2 DNA extract 2 using the Illumina DNA sample prep kit (PE-102-1001). We followed the manufacturer’s recommendations (San Diego, CA, USA), except for modifications described elsewhere [11 (link)] that were introduced for the purpose of analyzing ancient DNA. Briefly, blunt-end DNA fragments were produced using T4 and Klenow DNA polymerases, and 5’ ends were phosphorylated using T4 polynucleotide kinase. A deoxyadenosine was added to the 3’ ends of the fragments using Klenow exo- DNA polymerase and dATP to prevent the DNA fragments from ligating to one another during the adapter ligation reaction. Ligation to Illumina adapters was then performed using DNA ligase and one tenth of the amount of adapters recommended by the manufacturer.
Amplification of the library was performed using Phusion DNA polymerase. Twelve PCR cycles were enough to produce sufficient amounts of DNA for sequencing.
DNA sequencing was performed at Genoscope (Evry, France) on Illumina HiSeq 2000 (3 sequencing lanes) and HiSeq 2500 platforms (4 sequencing lanes) using HiSeq v3 chemistry with a read length set to 101 nucleotides and analysis on the single read mode.
Amplification of the library was performed using Phusion DNA polymerase. Twelve PCR cycles were enough to produce sufficient amounts of DNA for sequencing.
DNA sequencing was performed at Genoscope (Evry, France) on Illumina HiSeq 2000 (3 sequencing lanes) and HiSeq 2500 platforms (4 sequencing lanes) using HiSeq v3 chemistry with a read length set to 101 nucleotides and analysis on the single read mode.
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