Metagenomic sequencing of human wound tissue: invasive amebiasis
DNA libraries were prepared using TruSeqŽ Nano DNA Sample Preparation kits (Illumina) employing the low sample (LS) protocol. Briefly, 100 ng of each genomic DNA from the samples was fragmented by Adaptive Focused AcousticsTM Technology (Covairs, Inc., Woburn, Massachusetts, USA) using a Covaris M220 with settings for fragment sizes in the 350 bp range (duty factor 20%, peak incident power 50W, cycle per burst: 200, duration: 65 sec., temperature: 65°C). Fragmented chromosomal DNA was cleaned up with bead technology. End repair was performed according to the TruSeq protocols. Further clean up aqnd size selection was done with bead technology. 3'ends were adenylated, Illumina adapters were ligated and DNA fragments were enriched. The sizing and quantification of DNA fragments was determined using Agilent DNA 7500 kits (Agilent Technologies, Inc., Santa Clara, CA, USA) as described by the manufacture and Qubit (DNA BR Kit). Each individual library was adjusted to 4nM stock solution and of these 6pM were used for each individual sequence run. Sequencing was performed using Reagent Kit MiSeqŽ v3 (600 cycle) runs (Illumina), wherein a complete v3 run was used per sample.