Testing an rRNA subtraction method for RNA sequencing

Production of the initial library above did not include an rRNA subtraction step. This allowed for a faster library production workflow, with fewer steps and less handling of the sample, alleviating concerns about contamination of degradation- prone RNA becoming likely during a longer process. On the other hand, the fact that a polyA tailing step needs to be employed as part of the MessageAmp protocol when working with bacterial RNA means that all the RNA in the sample will have been polyadenylated including rRNA sequences. Since only mRNA is of interest here, it was decided to investigate the use of a rRNA-subtraction method to remove unwanted rRNA sequences prior to polyA tailing of the sample and subsequent amplification. The method chosen was a custom protocol described by Stewart et al

(2010), rather than a commercial kit-based method so that the probes used for rRNA subtraction would be sample-specific. The steps involved in metatranscriptome library preparation with and without rRNA subtraction are summarised in Fig 4.2.

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Figure 4.2 Comparison of total community RNA processing: A, without rRNA subtraction and B, with subtraction using the method of Stewart et al. (2010). Other

rRNA subtraction methods exist, using probes or other enzymatic means to selectively remove rRNA sequences but all are conceptually similar in that they are designed to remove as great a proportion of rRNA as possible in order to yield a final

product comprising mostly mRNA sequences for those applications focussing on active transcripts and expressed genes.

4.2.5.1 Method

Fig 4.3 illustrates the workflow for producing custom rRNA probes according to the method of Stewart et al. (2010). Briefly, this method requires DNA and RNA to be co- extracted from the same sample. The DNA is then used to produce biotinylated probe sequences that are in turn used to hybridise to the rRNA sequences in the total community rRNA sample and retrieve them using streptavidin- coated magnetic beads and magnetic separation.

Universal 16S and 23S rRNA gene primers, where one primer from each set contains a T7 promoter site, were used to amplify the almost full-length of the ribosomal genes from total community DNA. This amplification was carried out using the Herculase polymerase (Section 2.3.3) and the Eub16S and Eub23S primer sets (Table 2.2). This yields amplicons corresponding to the 16S and 23S rRNA sequences in the sample, with a T7 promoter site incorporated. This enables the amplicons to be used as templates for the MEGAscript high yield transcription kit, which allows the production of biotin-labelled anti-sense RNA probes, the sense being determined by how the T7 promoter site was incorporated during the initial PCR. The reaction conditions for probe production are listed in Table 4.3. The reaction was carried out at 37oC for 6 h.

Table 4.3 Megascript reaction components.

Component Quantity

PCR amplicons of rRNA genes 1 µl (approx. 500ng)

ATP (75 mM) 2 µl GTP (75 mM) 2 µl CTP (75 mM) 1.5 µl UTP (75 mM) 1.5 µl Biotin-11-CTP (10mM) 3.75 µl Biotin-16-UTP (10mM) 3.75 µl 10x Reaction Buffer 2 µl T7 RNA polymerase 0.5 µl

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Figure 4.3: Summary of the rRNA probe-based subtractive hybridisation method described by Stewart et al. (2010).

Biotinylated probes can then be incubated with the RNA sample to allow hybridisation to the rRNA sequences. which can the be removed via streptavidin coated magnetic beads. The hybridised RNA sample is transferred to beads aliquoted into microcentrifuge tubes placed in a magnetic rack. The hybridised rRNA probe complexes are captured by the beads which are in turn captured by the magnetic rack and remain adhered to the side of the tube. After a short incubation of a few min at room temperature, the remaining RNA sample can be removed by careful pipetting, now subtracted of its rRNA component.

4.2.5.2 Results.

The rRNA-subtracted library was to be made from the same RNA stocks as the initial non –subtracted library. DNA for PCR template material was taken from freezer stocks that contained DNA extracted at the same time as the RNA stock was produced. The DNA had been maintained at -80oC prior to use. PCR amplification was carried out to generate universal 16S and 23S rRNA gene sequences from the metagenomic DNA. 16S rRNA gene products of the expected size were generated in sufficient quantity. 16S rRNA probes were successfully made from the 16S rRNA gene amplicons using the MEGAscript kit.

Amplification of 23S ribosomal gene sequences from the sample DNA did not yield a single product of the expected size and therefore was not able to generate amplicons that could be used to produce rRNA probe sequences. Amplification of a discrete product band of the expected size for the 23S rRNA gene primers was achieved using E. coli genomic DNA. Repeated attempts to amplify a 23S rRNA gene product from the environmental sample were however never successful.

The 16S rRNA probes were stored at -80oC but ultimately were not used for rRNA subtraction as it was decided that the lack of accompanying 23S probes rendered this whole approach somewhat ineffective. Alternative methods of rRNA subtraction were available and therefore it was decided to employ one method in its entirety. The 16S rRNA probes could have been used to achieve some rRNA removal in addition to another method but introducing extra processing steps always risks damage to the RNA sample so use of these sample specific probes as an approach to ribosomal RNA depletion was abandoned.

4.2.6 Results of 454 sequencing a cDNA library produced from a polyadenylated and