- This protocol was developed for use with not-so-random
(NSR) primers. NSR primers can be designed for your organ-
ism(s) of interest by starting with a random hexamer pool and
doing an in silico analysis to identify primers predicted to
anneal to the 5S, 16S, and 23S ribosomal RNA sequences in
your genome(s) of interest. This is done by computationally
aligning all possible hexamers (4096) to the desired 23S, 16S,
and 5S rRNA sequences. Hexamers with a perfect match
should be excluded from the NSR primer collection, as
descried originally by Armour et al. [2]. Individual NSR primer
sets have been created for bacteria including: relatedBurkhol-
deriaspecies [4, 9],Pseudomonas aeruginosa[10], andRho-
dopseudomonas palustris[11]. If one plans to generate de novo
an NSR primer set, the primers should be ordered individually
(likely in a 96 multi-well format) and pooled as desired.
Though every primer set will vary, do not be alarmed if the
NSR primer set comprises vastly fewer primers than the starting
random hexamer pool. ForBurkholderia, the NSR primer set
included 949 primers. An NSR primer set can also be designed
for a group of related organisms, as the rRNA sequences will be
similar. For example, one primer set was developed for use with
threeBurkholderiaspecies:B. thailandensis,B. pseudomallei,
andB. mallei. Creating a new NSR primer set is costly and
time-intensive, so it may be a good idea to investigate if an NSR
primer set has already been developed for an organism moder-
ately related to one’s organism of choice. For example, it is
reasonable to assume theBurkholderiaNSR primer set could
be used on other more distantly relatedBurkholderiaspecies.
Additionally, the NSR primer set used forR. palustriswas
successfully used for RNAseq onBradyrhizobium japonicum
(Nathan Ahlgren, Caroline Harwood, and E. Pete Greenberg,
personal communication). A final alternative for NSR priming
is to purchase a commercially available primer set. There are
now “universal” NSR primer sets that work very well for
groups of related organisms (a product line is available through
NuGEN). Regardless of the approach taken, it is prudent to
perform a test RNAseq run to ensure that the NSR primer set
efficiently limits rRNA amplification, while retaining good cov-
erage for the rest of the genome. TheBurkholderiaNSR primer
set achieved an average coverage of 87–106 hits per gene,
depending on the species.
If you choose not to use NSR primers, you will need to
deplete rRNA from your total RNA sample. The protocol could
then be adapted to use random hexamers for cDNA synthesis. - If multiplexing (running more than one sample per lane in the
Illumina Sequencer) is desired, it is at this stage that a barcode
is introduced. Only the forward adapters (FwdAD) are
190 Charlotte D. Majerczyk