ena-STUDY-irena.maus-09-04-2018-11:53:12:217-2913
Methods for the extraction of bacterial DNA
Choosing an appropriate DNA extraction method for determination of bacterial diversity is crucial and can be a challenge in case that preliminary knowledge on the sample matrix to be analyzed is scarce or lacking. The aim of the present study was to determine a DNA extraction kit suitable for 16S rRNA gene amplicon sequencing of chicken caecum taking into account criterion such as high technical reproducibility and bacterial diversity as well as easy handling. The protocols tested differed strongly with respect to DNA concentrations, its purity, technical reproducibility and furthermore alpha and beta diversity. Differences in relative taxa abundances due to the DNA extraction conditions applied were detected. Some protocols showed under-represented amount of Ruminococcaceae, Ruminococcaceae insertae sedis, Defluviitaleaceae and Erysipelotrichaceae sequences in their taxonomic profiles, which belong to the common caecal community members. Furthermore, protocols consisting of a cleaning step to remove PCR inhibitors led to significantly increased technical reproducibility of the DNA extraction as well as alpha diversity. The removal of PCR inhibitors prior to 16S rRNA gene amplicon PCR proved to be one of the main drivers for high technical reproducibility as well as a high bacterial diversity within the chicken caecum bacterial community.
Choosing an appropriate DNA extraction method for determination of bacterial diversity is crucial and can be a challenge in case that preliminary knowledge on the sample matrix to be analyzed is scarce or lacking. The aim of the present study was to determine a DNA extraction kit suitable for 16S rRNA gene amplicon sequencing of chicken caecum taking into account criterion such as high technical reproducibility and bacterial diversity as well as easy handling. The protocols tested differed strongly with respect to DNA concentrations, its purity, technical reproducibility and furthermore alpha and beta diversity. Differences in relative taxa abundances due to the DNA extraction conditions applied were detected. Some protocols showed under-represented amount of Ruminococcaceae, Ruminococcaceae insertae sedis, Defluviitaleaceae and Erysipelotrichaceae sequences in their taxonomic profiles, which belong to the common caecal community members. Furthermore, protocols consisting of a cleaning step to remove PCR inhibitors led to significantly increased technical reproducibility of the DNA extraction as well as alpha diversity. The removal of PCR inhibitors prior to 16S rRNA gene amplicon PCR proved to be one of the main drivers for high technical reproducibility as well as a high bacterial diversity within the chicken caecum bacterial community.