Abstract
With the rapid development of high-speed DNA sequencing technologies, it became feasible to sequence deeply into cDNA libraries prepared from RNA samples. Such cDNA libraries can benefit from the development of full-length cDNA cloning technologies providing means to obtain sequence information on the entire RNA transcripts or their selected 5′ end. Comprehensive overviews on transcriptomes can be obtained today by combination of those new sequencing technologies with large-scale cDNA library preparation forming the basis to different approaches for transcriptome profiling.
In this chapter, we describe the use of full-length cDNA preparations in combination with shotgun sequencing in mRNA profiling (so-called RNA-Seq methods for “RNA sequencing”) and RNA-Seq profiling starting directly from RNA. Moreover, we describe the use of cap analysis gene expression (CAGE) for high-throughput mRNA detection and determination of transcription start sites (TSS) on the genome level. Here we applied “nanoCAGE", which uses template switching in the 5′ end selection step, to obtain CAGE data from very small amounts of RNA. We compare the sequencing data obtained by the three different library preparation methods and give directions for a bioinformatics pipeline used for their analysis. Examples are taken from our studies on the transcriptional regulation of gene expression during behavioral maturation of worker honey bees (Apis mellifera) to advise on transcriptome profiling strategies.
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Abbreviations
- CAGE:
-
Cap analysis gene expression
- CPCC:
-
Cophenetic correlation coefficient
- DEGs:
-
Differentially expressed genes
- EST:
-
Expressed sequence tag
- FPKM:
-
Fragments per kilobase per million reads
- GO:
-
Gene ontology
- MAPQ:
-
Mapping quality
- RNA-Seq:
-
RNA sequencing
- RPKM:
-
Reads per kilobase per million
- TMM:
-
Trimmed mean of M-values
- TPM:
-
Tags per million
- TSSs:
-
Transcription start sites
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Acknowledgment
We want to express our great thanks to Adam R. Hamilton, Yulia A. Medvedeva, Tanvir Alam, Intikhab Alam, Magbubah Essack, Boris Umylny, Boris R. Jankovic, Nicholas L. Naeger, Makoto Suzuki, and Gene E. Robinson for their great support for our honey bee project, which would have not been possible without working together with them. We further want to thank Charles Plessy and Piero Carninci for their support and encouragement for using CAGE.
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Khamis, A.M., Bajic, V.B., Harbers, M. (2016). Transcriptome Profiling Strategies. In: Aransay, A., Lavín Trueba, J. (eds) Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing. Springer, Cham. https://doi.org/10.1007/978-3-319-31350-4_4
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