title: Shotgun sequencing of Homo sapiens: healthy female infant
design: The samples were homogenate using bead beater (Biospec Products, USA). The mixture contained 100 mg of 150 to 212 m glass beads (Sigma, USA) in final volume of 10% chloroform in 1 ml of phosphate-buffered saline (PBS). After homogenization, the samples were centrifuged at 2000 x g to remove large debris, and the supernatants were filtered in Spin-X 0.45 m pore filters (Costar, NY) at 10,000 x g for 10 to 20 min. 400 l of filtered samples was treated with Turbo DNase (4U) (Ambion, USA) and RNAse (2U) (Sigma, USA) for 30 min at 37oC, and nucleic acids were extracted using the PureLink Viral RNA/DNA extraction kit (Invitrogen, USA). The extracted nucleic acids were eluted in nuclease-free water, aliquoted, and stored at -70oC until further use. Later, the RNA was revere transcribed using SuperScript III reverse transcriptase (Invitrogen, USA). The second strand DNA was generated by two consecutive rounds of synthesis using Sequenase 2.0 (USB, USA). The obtained DNA was amplified using Phusion High fidelity polymerase (Finnzymes). The amplified DNA was cleaned from oligos and nucleotides by ZYMO DNA Clean & Concentration-5 kit. Sequencing libraries were prepared using a Nextera XT DNA library preparation kit (Illumina). Samples were tagged, multiplexed and sequencing was performed by single-end 72 cycles of nucleotide extension in a NextSeq500 Illumina platform.