LIBRARY PREPARATION
During the process of library preparation, DNA or RNA molecules are selected, amplified or modified in order to be detected by the sequencing platform. Particularly in meta-omic studies, where multiple samples are sequenced simultaneously, these molecules are also tagged by molecular barcodes so that we can track each DNA/RNA molecule to their original samples. This process differs between DNA and RNA samples.
DNA genomic library
OmiBox makes use of Oxford Nanopore Technologies for bacterial taxonomic analyses (16S Barcoding Kit 1-24; SQK-16S024). For this purpose, 16S rDNA gene is targeted and amplified by using specific primers and PCR amplification as shown in the figure below. At the end of this process, a collection of 16S rDNA amplicons will be ready to be loaded onto the MinION sequencer.
RNA genomic library
RNA sequencing is commonly used for meta-transcriptomic studies where the mRNA fraction is analysed to examine the cellular function of entire microbial communities. Although Oxford Nanopore Technologies offers the direct sequencing of RNA molecules, the amounts of mRNA needed usually exceed the concentrations contained in environmental samples, particularly in marine waters. But researcher came up with an ingenious idea: the synthesis of cDNA (copy DNA) by the retrotranscription of the mRNA molecules. By doing so, cDNA can then be amplified by a regular PCR and thus the quantity of cDNA is increased exponentially. From there, the creation of a cDNA library follows basically the same steps as a regular DNA library (see figure below).
However, making a genomic library from the original RNA pool is trickier than it seems. To begin with, all DNA remaining after the extraction of RNA must be significantly depleted (practically to none) to avoid the amplification of genomic DNA during the cDNA synthesis. This is usually done by treating the RNA sample with DNase enzymes (see Zymo DNA/RNA extraction protocols).
More importantly, only mRNA is meant to be analysed, so we must get rid of all the other RNA species, particularly rRNA, which is (by far) the most abundant RNA in the cells. This is a time-consuming process that may take up to several hours. In OmiBox, we make use of the Pan-Prokaryote riboPOOL kit provided by siTOOs BIOTECH.
