Starter Guide to mRNA Sequencing (Novogene)
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By Ayush Desai
- RNA sequencing (RNA-seq) offers unprecedented insight into genomics
- mRNAs are captured and converted to cDNA for library prep
- RNA-seq, a high-throughput NGS technique, reveals gene expression profiles and continuous transcriptome variations
What is mRNA Sequencing?
Important factors in mRNA sequencing
- single vs paired end
- stranded vs paired end
- sequencing length
- coverage
Why Choose mRNA Sequencing?
RNA (Ribonucleic acid)
Role
- Single stranded
- Three base codon -> corresponding amino acid
- Production of proteins through translation
- Cellular functionality
- Coding region
- RNA splicing becomes important
Carries the coding sequences for protein synthesis and are called transcripts
Why mRNA Sequencing?
1. Annotation
2. Quantification
3. Target Prediction (ncRNA)
Applications of mRNA Sequencing
- Quantitative profiling of transcripts in diverse tissues, conditions, and treatments
- Uncovering novel transcripts, alternative splicing, and variations
- Studying developmental mechanisms, drug resistance, and biomarkers through tissue-specific transcripts
- Integrating omics analysis with the transcriptome
- Investigating pathogenic mechanisms and clinical subtypes
Applications of RNA Sequencing
Treatment vs Control
- Compare different samples under different conditions
- Control group and treatment group
Treatment vs Control
Tissue-specific Gene Expression
- Why can we see with our eyes?
- Why is there a high localization of hair in certain areas?
- Alopecia Areata
- Autoimmune condition
- Cortisol injections (temporary)
- JAK inhbitor
Tissue Specfic Gene Expression
https://www.medicalnewstoday.com/articles/70957
Time Course Gene Expression
Time Course Gene Expression
- Impact of new medicine dose on gene profile
- Consider pairing with ncRNA sequencing for optimal results
- Also making sure the time is sufficient for results
- Saccharomyces cerevisiae (Brewer's Yeast)
mRNA Technologies and Platforms
Technologies for mRNA Sequencing
NovaSeq X Plus
- Long read sequencing
- Short read sequencing
- mRNA
- ncRNA
NovaSeq X Plus
NovaSeq 6000
- Short read sequencing
- mRNA
- ncRNA
NovaSeq 6000
PacBio & Nanospore
- Better for long reads
- Better for isoform characterization
- Target RNA: Isoseq & Nanospore RNA seq
PacBio & Nanospore
Nanospore PromethION Platform
RNA Sequencing Workflow
Library Quality Control
Sample Quality Check
Data Quality Control
- Sequencing quality score of given base Q, is defined by equation below
- e represents the estimate probability of base cell being incorrect
Require a minimum of 0.4 micrograms of total RNA for eukaryotic project
1. Agarose gel electrophoresis
2. Nanodrop reading
3. Agilent Machine
mRNA Sequencing Workflow
Problem: What if there is not enough RNA?
Solution: Smart sig ultra-low input RNA kit
Sample Preparation
Methods
- Trizol method
- RNA extraction kits (commercially available)
- Ship with Dry Ice
Design the Analysis
1. Name your samples and groups
2. Chose your reference genome
3. Design your comparsions
Analysis
Name Your Samples and Groups
Samples and Groups
Design Your Analysis
Reference Genome
Chose reference genome
- Novogene database
- Provide your own database
- Another database
- NCBI
- Ensembl
Design Your Comparsions
Design Comparsions
- Fill in the treatment and control
- Make sure to have a clear understanding of your research purpose
- Venn diagram
https://bioinfo.iric.ca/venn-diagrams-a-visualization-nightmare/
Results Overview
1. Raw data
2. Mapping
3. Differential expression analysis
Results
Raw Data
1. Raw Data
https://www.researchgate.net/figure/FASTQ-file-1st-line-always-starts-with-the-symbol-followed-by-the-sequence_fig8_252323690
Mapping
- Results provided in excel format
- Good for mapping that will show data
- Pie chart will show different types of reads
2. Mapping
Differential Expression Analysis
- Volcano plots
- Good to see large fold changes
- Most biologically significant genes
3. Differential Expression Analysis
https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/rna-seq-viz-with-volcanoplot/tutorial.html