Transcriptome Sequencing – Applications and Preparation

One of the most common uses for RNA Sequencing (RNA-Seq) is gene expression analysis (sometimes referred to as mRNA-Seq). However, there are many other types of RNA-Seq that can be used to address various research questions. Some other types of RNA-Seq include whole transcriptome sequencing, small RNA-Seq, single cell RNA-Seq and low-input RNA-Seq.

The type of RNA-Seq that should be chosen is directly related to the project end goal. Different project goals may call for the output of different information about the transcriptome.

The use of various library preparation protocols and sequencing runs will determine the type of data output. These differences are not minute and choosing the wrong protocol can lead to disastrous outcomes. For instance, if you need information about the whole transcriptome, including alternative splice variants and small RNAs, but utilize an mRNA-Seq library prep protocol (standard poly-A selection) you may lose vital strand-specific information or all content about small non-coding RNAs.

In order to help you avoid unfavorable sequencing results, I’d like to review some RNA-Seq applications and the suggested library preparation protocols.

mRNA-Seq

Differential gene expression analysis of known coding transcripts is by far the most common application of RNA-Seq. mRNA-Seq offers insight into the expression of mRNAs or protein-coding regions of the genome.

Real world applications of mRNA-Seq

Some of the ways that this type of RNA-Sequencing can be used includes:

• Drug development: For instance, you may want use this method if you are interested in determining the effects of drug treatment on protein coding regions of the transcriptome.
• Cancer Research: Identifying cancer causing regions of the genome is an important research goal. Many abnormal splicing events have been implicated in cancer progression or development. Monitoring gene expression changes can help researchers identify cancer-causing genes or regions of the transcriptome that are responsive to treatment.

Suggested library preparation protocol and sequencing run type:

Standard poly-A selection library preparation protocols and a paired-end run on the Illumina HiSeq platform generating 20-30 million reads per sample is suggested for mRNA analysis. Standard RNA library preparation techniques may or may not include strand-specific information. Strand-specific information is important to retain the orientation of the original RNA transcript. Contact us to determine whether strand-specific library preparation protocols are right for your experiment.

Whole Transcriptome Sequencing

Abundant information about the whole transcriptome is one of the great perks of using next generation sequencing. Whole transcriptome sequencing can be used to gain information about long non-coding transcripts, isoforms, alternative splice variants and novel transcripts. It may even be used for genomic variant detection.

Real world applications of whole transcriptome sequencing

It is sometimes the case that you need more information outside of just the coding portion of the transcriptome. Whole transcriptome sequencing offers insight into both coding and non-coding transcripts.

• Complex human disease analysis: Certain diseases may be influenced by a combination of factors which are both genetic and environmental. These factors lead to changes in multiple genes and both pre- and post-transcriptional changes may need to be analyzed. Whole transcriptome sequencing can be used to assess how changes are made in both mRNA and non-coding RNAs.
• Cancer research: There is increased interest in identifying how non-coding regions of the transcriptome contribute to cancer progression. For example, long intergenic non-coding RNAs (lincRNA) are affiliated with protein-chromatin interactions. Dysregulation of lincRNA is thought to contribute to tumor growth, with differential regulation of these RNAs being observed between normal and metastatic tissues¹. Whole transcriptome sequencing can be used to characterize novel instances of lincRNAs and may contribute to important areas of cancer research.
• De novo transcriptome assembly: Reference genomes for many organisms are still poorly annotated or do not exist. Whole transcriptome sequencing may be utilized for de novo assembly and gaining important information about the transcriptomes of non-model organisms.

Suggested library preparation protocol and sequencing run type:

For whole transcriptome sequencing, it is important to retain as much information about the transcriptome as possible and strand-specific RNA library preparations are suggested.

In addition, it is advised that for whole transcriptome sequencing, you utilize rRNA depletion protocols instead of poly-A selection. It is often the case that alternative splice variants do not retain polyadenylated tails and thus poly-A selection can lead to the loss of transcript information. The suggested depth of coverage for this type of experiment will have a broad range of 30-100 million reads per sample and is dependent on the annotation of the organism as well as the specific goal of the experiment. For well-annotated organisms it may be the case that lower coverage is needed; however, for variant detection increased depth will be necessary for SNP analysis of genes with low expression.

Small RNA-Seq

Information about the epigenome can be obtained through small RNA-Seq. Small RNAs such as miRNAs, siRNAs, piRNAs, etc. are important regulators of gene expression.

Real world applications of small RNA-Seq

A couple of examples of how epigenetic modification is important to research are listed below:

• Cancer Research: Posttranscriptional regulation of gene expression has been shown to play a role in lung cancer progression². Using small RNA-Seq, researchers at Aarhus University observed differential regulation of both miRNA and piRNA in metastasizing and non-metastasizing cancer samples. These findings may be important potential cancer biomarkers for future points of study.
• Complex disease analysis: Posttranscriptional gene expression regulation contributes to many complex human diseases. Information about small non-coding RNAs is often used in conjunction with mRNA-Seq or whole transcriptome sequencing and gain an understanding of complex disease regulation.

Suggested library preparation protocol and sequencing run type:

For small RNA-Seq the choice of RNA extraction protocol is especially important because you want to make sure to retain all small RNAs. Many kits are designed to optimize output of high-quality RNA by providing steps that remove impurities or degraded RNA products. However, it is sometimes the case that this additional step also removes many small RNAs. We recommend the extraction of total RNA using TRIzol. If you use a kit, make sure the kit is specific to the retention of small RNAs.

Small RNA library preparation and a single-end or paired-end run (50 base pair read length) on the Illumina HiSeq platform, generating anywhere between 5-15 million reads per sample is adequate for most downstream data analysis purposes.

Single-Cell RNA-Seq

Single cell RNA-Seq (scRNA-Seq) can be used to identify heterogeneity of transcriptomes from cellular populations.

Real world applications for single-cell RNA-Seq

Single-cell RNA-Seq (scRNA-Seq) may be utilized by researchers interested in the analysis of heterogeneous stem cell populations.

• Stem cell research: An RNA-Seq Blog entry³ reviewed an article published in Nature which utilized single cell sequencing to analyze intestinal stem cell self-renewal pathways. In this study, researchers from Stanford University utilized scRNA-Seq in order to gather information about cellular subtypes in the intestine.

Suggested library preparation protocol and sequencing run type:

Phalanx Biotech offers single cell sequencing options on the 10X Chromium platform⁴. This platform gathers gene expression information on a cell-by-cell basis. For more information about scRNA-Seq please contact us.

Summary of RNA-Seq Applications

There are various ways that RNA-Seq data can be utilized. Understanding the various applications and the differences in preparation methods may help you obtain the appropriate sequencing data output.

Various types of RNA-Seq have been utilized by pharmaceutical companies, clinical research organizations, research universities, etc. These studies have shown that RNA-Seq is a vital tool that contributes to our understanding of both pre- and post-transcriptional regulation of gene expression.

Contact us at info@onearray.com or call 619-568-2999 ext. 111 to speak with an expert about your next RNA-Seq experiment!

 

Sources

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057914/
2. http://www.rna-seqblog.com/small-rna-sequencing-reveals-metastasis-related-micrornas-in-lung-adenocarcinoma/
3. http://www.rna-seqblog.com/scrna-seq-unravels-a-novel-pathway-for-stem-cell-self-renewal/
4. https://www.10xgenomics.com/news/10x-genomics-new-chromium-system-enables-full-access-to-critical-molecular-and-cellular-information/