ena-STUDY-FISABIO-29-01-2019-09:41:14:013-26
Comparison of five DNA extraction and purification methods for microbiome characterization from the lung tissue.
A total of 7 individuals (four children and three adults) selected on the basis of unexpected death were included in this study.For each autopsied subject, the right upper lobe was removed, small samples were obtained from deep lung tissue, cut into small pieces, divided in five aliquots (0.4 g) and frozen at -80ºC for DNA extraction. Five DNA extraction protocols were applied on each piece o sample: protocol 1, using the QIAamp DNA Mini kit (Qiagen); adding a bead-beating step (protocol 2); a phenol-chloroform step (protocol 3); both bead-beating and phenol-chloroform steps (protocol 4); and a pre-treatment step followed by the bead-beating and phenol-chloroform steps (protocol 5).For all samples, the 16S rRNA gene analysis for the bacteria and the internal transcribed spacer (ITS) region for fungal community were carried out, respectively. PCR amplification of 469  bp of the V3-V4 regions in the case of bacteria, and  spanning about 289 bp of the small subunit and the 5.8S region of the rRNA operon in the case of fungi were performed and sequencing of the amplicons using the Illumina MiSeq with 300 bp x2 chemistry.
A total of 7 individuals (four children and three adults) selected on the basis of unexpected death were included in this study.For each autopsied subject, the right upper lobe was removed, small samples were obtained from deep lung tissue, cut into small pieces, divided in five aliquots (0.4 g) and frozen at -80ºC for DNA extraction. Five DNA extraction protocols were applied on each piece o sample: protocol 1, using the QIAamp DNA Mini kit (Qiagen); adding a bead-beating step (protocol 2); a phenol-chloroform step (protocol 3); both bead-beating and phenol-chloroform steps (protocol 4); and a pre-treatment step followed by the bead-beating and phenol-chloroform steps (protocol 5).For all samples, the 16S rRNA gene analysis for the bacteria and the internal transcribed spacer (ITS) region for fungal community were carried out, respectively. PCR amplification of 469  bp of the V3-V4 regions in the case of bacteria, and  spanning about 289 bp of the small subunit and the 5.8S region of the rRNA operon in the case of fungi were performed and sequencing of the amplicons using the Illumina MiSeq with 300 bp x2 chemistry.