Oxford Nanopore MinION sequencing of multiplex PCR amplified, pathogen-spiked blood and plasma samples
Oxford Nanopore MinION sequencing of multiplex PCR amplified, pathogen-spiked blood and plasma samples
Human blood or plasma samples from anonymous donors are spiked with pathogen stocks in the laboratory Nucleic acids from plasma spiked with viral pathogens were extracted with the QIAamp MinElute Virus Spin kit (Qiagen, Valencia, CA) and DNA from bacteria or protozoan parasite spiked whole blood specimens was extracted with the DNA Blood Mini kit (QIAamp, Qiagen,) according to the manufacturers' protocols PCR Amplification Nucleic acid samples were added to a reverse transcription (RT) reaction. The products from the RT reaction were divided among four PCR reactions each containing one of the primer pools with internal positive controls and the primer-linker oligonucleotide. After several rounds of specific amplification, the primer-linker continues the amplification of the existing PCR products. The PCR products were combined and purified with a DNA clean-up system (Wizard SV Gel and PCR Clean-Up System, A9282, Promega, Madison, WI). PCR product samples were used to prepare a library with the Oxford Nanopore Ligation sequencing amplicons - native barcoding kit (SQKNBD112.24) following the manufacturer's instructions.