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Starter Guide to mRNA Sequencing (Novogene)

Thank You for Listening!

Please let me know if you have any questions!

By Ayush Desai

mRNA Sequencing

  • 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

biostars.org/

RNA

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

mRNA

Carries the coding sequences for protein synthesis and are called transcripts

Why mRNA Sequencing?

Polozo and Lee, 2015

1. Annotation

  • What are they?

2. Quantification

  • How many of each?

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

theyenews.com

  • 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

Alopecia Areata

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)

BMC Bioinformatics

mRNA Technologies and Platforms

What Type of RNA?

Technologies for mRNA Sequencing

Pros and Cons

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

Pacbio REVIO Platform

  • 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

  • Q = -10log10(e)

  • 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

Library Construction

Bioinformatics Analysis

Sample Preparation

Sequencing

Methods

- Trizol method

- RNA extraction kits (commercially available)

- Ship with Dry Ice

(David Corney 2013)

Design the Analysis

1. Name your samples and groups

2. Chose your reference genome

3. Design your comparsions

Analysis

Step 1

Name Your Samples and Groups

Samples and Groups

Step 2

Design Your Analysis

Reference Genome

Chose reference genome

  • Novogene database
  • Provide your own database
  • Another database
  • NCBI
  • Ensembl

Step 3

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

FASTQ format

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

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