Organism-specific rRNA capture system for application in next-generation sequencing.
RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA) and transfer RNA (tRNA) have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA) when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.
Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR.
Sai-Kam
SK
Li
Jun-Wei
JW
Zhou
Aldrin Kay-Yuen
AK
Yim
Alden King-Yung
AK
Leung
Stephen Kwok-Wing
SK
Tsui
Ting-Fung
TF
Chan
Terrence Chi-Kong
TC
Lau
D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D020102:Mycobacterium smegmatis / Q000235:genetics*; D015347:RNA Probes / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity
D015347:RNA Probes; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012335:RNA, Ribosomal