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Notwithstanding The variability of methods which have been launched to beat artefacts linked to details compositionality, they have got not been adopted to the human microbiome investigation. Of Notice, none of the clinical trials posted in the course of the previous year (other than those specifically addressing the compositionality problem [five, six]) utilized quantitative microbiome profiling. In this article, we existing how quantitative PCR (qPCR)-dependent bacterial enumeration might be integrated to NGS pipelines to provide a possible approach to estimate absolute abundances from NGS knowledge, and therefore promote the use of quantitative microbiome profiling in the sphere of human microbiome.
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Given that numerous common bacterial primers are actually developed and optimized specifically for qPCR or NGS, it is highly recommended to think about probable biases ensuing from primer-particular amplification effectiveness for certain taxa [35] and also differential primer protection, when applying unique primer sets for qPCR and NGS. Within this analyze, we chose a widely-utilized universal bacterial primer set optimized for qPCR [29] that in the same way targets the V3-V4 hypervariable regions given that the primer set employed for Illumina MiSeq (S1A Table).
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