For 16s rDNA metagenomic analysis, do I need to do the following steps?

DNA extraction-> Purification-> PCR amplification-> Clone -> Pick every single colony-> PCR amplify -> Sequence -> Phylogenetic Analysis

For the abundance of a particular gene abundance, what steps do I need to follow? Is it necessary to clone?


  • 1
    $\begingroup$ 16S rDNA? You mean 16S ribosomal RNA, right? If yes then no you don't need to do a library in bacterial clones anymore. With next generation sequencing technologies the library is made In-Vitro followed by PCR-like step (e.g. Illumina Nextera XT kit). What you would do is RNA extraction -> Reverse-transcription -> PCR amplification -> library building using a kit -> Sequencing -> Phylogenetic analysis. $\endgroup$ – cagliari2005 Mar 26 '15 at 16:49

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