- Course overview
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- Introduction
- Real-time PCR
- Microarrays
- What is Next Generation DNA Sequencing?
- Biological interpretation of gene expression data
- Genotyping, epigenetic and DNA/RNA-protein interaction methods
- DNA/RNA-protein interactions
- Summary
- Quiz: Check your learning
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- References
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Design considerations
When we design an RNA-seq experiment we have to consider the following aspects (9):
- library construction
- sequencing depth or library size
- number of replicates
Library preparation
Library preparation methods vary depending on the type of RNA being used, whether strand specificity is necessary and the type of reads.
One important aspect is choosing which population of RNA will be converted to a cDNA library. Using total RNA allows detection of non-coding as well as mRNAs, but may require additional enrichment steps (e.g. ribosomal RNA depletion) to allow the detection of transcripts of low abundance. PolyA+ RNA enrichment can be used to purify mRNAs and is useful in studies of eukaryotic organisms.
Another consideration is whether to generate a strand-specific library that retains the orientation of the original RNA transcript, which may be critical to identify antisense or non-coding RNA.
Furthermore, sequencing can involve single-end (SE) or paired-end (PE) reads. Paired-end sequencing means sequencing both ends of the cDNA fragments and aligning the forward and reverse reads as read pairs (Figure 13). Paired-end reads are preferable for de novo transcript discovery or isoforms expression analysis, as well as to characterise poorly annotated transcriptomes.
Sequencing depth or library size
Sequencing depth or library size refers to the number of sequenced reads for a given sample. As the sample is sequenced to a deeper level, the reads are likely to cover a larger proportion of the genome/transcriptome, allowing more transcripts to be detected with more precise quantification. Optimal sequencing depth depends on the aims of the experiment and on the complexity of the target transcriptome.