Modified bisulfite sequencing was optimized for rRNA, tRNA, and miRNA molecules from Drosophila.
Bisulfite treatment has been most widely used to detect dm5C (DNA m5C). The treatment essentially converts a cytosine to a uridine, but methylated cytosines would be unchanged by the treatment.
Previous attempts to develop m5C sequencing protocols using bisulfite treatment were not able to effectively address the problem of the harsh treatment of RNA which causes significant degradation of the molecules. Specifically, bisulfite deamination treatment (high pH) of RNA is detrimental to the stability of phosphodiester bonds. As a result, it is difficult to pre-enrich RNA molecules or to obtain enough PCR product of the correct size for deep sequencing.
A modified version of bisulfite sequencing was developed by Schaefer et al. (2009) which decreased the temperature at which bisulfite treatment of RNA from 95 °C to 60 °C. The rationale behind the modification was that since RNA, unlike DNA, is not double-stranded, but rather, consists of regions of single-strandedness, double-stranded stem structures and loops, it could be possible to unwind RNA at a much lower temperature. Indeed, RNA could be treated for 180 minutes at 60C without significant loss of PCR amplicons of the expected size. Deamination rates were determined to be 99% at 180min of treatment.
After bisulfite treatment of fragmented RNA, reverse transcription is performed, followed by PCR amplification of the cDNA products, and finally deep sequencing was done using the Roche 454 platform.
Since the developers of the method used the Roche platform, they also used GS Amplicon Variant Analyzer (Roche) for analyzing deep sequencing data to quantify sequence-specific cytosine content.
However, recent papers have suggested that the method have several flaws: (1) Incomplete conversion of regular cytosines in double-stranded regions of RNA; (2) areas containing other modifications that resulted in bisulfite-treatment resistance; and (3) sites containing potential false-positives due to (1) and (2)  In addition, it is possible the sequencing depth is still not high enough to correctly detect all methylated sites.