week 2: mRNA seq 1

Question 1

purpose

Answer 1

The purpose of this training will be to inform new hires of RNA-based sequencing services

Introduce the biological and commercial significance of RNA

Discuss the process of RNA sequencing projects from QC – data delivery

Build a foundational knowledge of RNA services offered by Novogene

Question 2

RNA vs. DNA

Answer 2

Molecular differences
- RNA: Adenine, guanine, uracil, cytosine
- DNA: adenine, guanine, thymine, cytosine

Strand #

DNA is relatively stable and only minor sample degradation is seen with shipping (barring any delays/complications)

RNA is highly UNSTABLE and sample degradation may be seen with shipping (especially if going to China)

Question 3

the central dogma of biology

Answer 3

DNA to RNA to mRNA to protein

DNA to RNA = transcription

RNA to mRNA = polyadenation

mRNA to protein = translation

Question 4

mRNA

Answer 4

– polyadenylated mRNA is RNA that is expressed and/or in the process of being expressed. It is therefore an indication of the downstream effects of modifications made to biological control systems that are influenced by both internal and external factors. (i.e. drugs, environmental factors, DNA modifications, etc.)

Question 5

mRNA vs. “Other” RNA

Answer 5

Polyadenylated mRNA is RNA that is expressed and/or in the process of being expressed. It is therefore an indication of the downstream effects of modifications made to biological control systems that are influenced by both internal and external factors. (i.e. drugs, environmental factors, DNA modifications, etc.)

Other types of RNA are important for many applications as well, however, a main focus within the genomics is on actively expressed transcripts, that is why mRNA is specifically so important

Question 6

TLDR

Answer 6

“Gene Expression” study = mRNA Seq

Key feature that differentiates mRNA from other RNA types = polyA tail

Question 7

Quality Control Steps

Answer 7

total RNA - sample quality control - library construction

library construction - library quality control - sequencing

sequencing - data quality control - bioinformatics analysis

Question 8

QC & Library Prep

Answer 8

Starting Material

Cell Pellets or Tissue / Whole Blood / FFPE
Novogene can extract the total RNA from most cell / tissue
Novogene can never guarantee the success of an extraction as it’s highly depended on the quality of the starting material
Extraction costs are $50-$70/sample (variable based on sample count)

Total RNA
>100ng of total RNA needed for non-directional library prep (most common)
>400ng of total RNA needed for directional library prep
What if the client has less than 100ng?? – we can accommodate it through one our low input pipelines

Question 9

Sample QC

Answer 9

QC Method = Agilent 2100 or AATI fragment analyzer

Checks RNA integrity, purity, and quantity

For all normal RNA/DNA pipelines, we allow for 2 library QC’s within the quoted cost. (i.e. 1 sample + 1 backup can be QC’d for no additional cost)
Any additional QC’s that may be needed on top of that

Question 10

RNA Sample Requirements

Answer 10

Client Facing Sample
Requirements

RIN on scale of 1-10, the closer to 10 the better

Question 11

RNA Sample Requirements

Answer 11

Qubit
Quantification of RNA
Unit: ng/µL
Not used as part of Novogene internal QC, but is a common tool used my clients.

A qubit fluorometer is a sensitive instrument used to quantify RNA (as well as DNA and protein) based on fluorescence
Fluorescent dye used in the Qubit assay has a high affinity for RNA and binds to it, forming a dye-RNA complex. The amount of light emitted is proportional to the
amount of RNA present.

Question 12

Nanodrop

Answer 12

Quantification & Qualification of RNA
Units: ng/µL & Absorbance Units (A)
Not used as part of Novogene internal QC, but is a common tool used my clients.

Sample Requirements:
OD260/280 ≥ 2.0
OD260/230 ≥ 2.0

260nm: nucleic acids (RNA and DNA)
Because DNA and RNA absorb at the same wavelength, DNA contamination can inflate concentrations
280nm: proteins
230nm: contaminants of Trizol, guanidine thiocyanate, EDTA, phenol and guanidine hydrochloride etc….

Question 13

Agilent 2100 Bioanalyzer / AATI Fragment analyzer / Tapestation

Answer 13

Quantification & Qualification of RNA

Agilent 2100 Bioanalyzer
Utilizes microfluidics technology to perform electrophoresis on a miniaturized scale.
Highly accurate, well known instrument, produces gel like images

AATI Fragment Analyzer
Capillary electrophoresis system that separates nucleic acids based on size and charge.
Suitable for integration into automated workflows for large-scale projects.
Used by Novogene

TapeStation
Automated electrophoresis system, samples are detected using fluorescence
Moderate resolution compared to Bioanalyzer.

Question 14

Library Prep: PolyA Selection vs rRNA Depletion

Answer 14

Poly-A tail selection: oligo(dT) beads selectively pull down the mRNA, leaving everything else behind

rRNA depletion: rRNA is selectively depleted from the sample. In the prep step, tRNA and sRNA aren’t captured due to small size. Leaving mRNA, lncRNA, and circRNA primarily to be captured in the prep

Question 15

rRNA-Depletion uses

Answer 15

When other ncRNAs are of interest

Eukaryotic spp from FFPE

For all Prokaryotic spp

Question 16

Library Prep: Directional vs Non-Directional Prep

Answer 16

Directional = Stranded = Strand Specific
Non-Directional = Non-Stranded

What: Directional mRNA library preparation preserves RNA strand directionality (3’ -5’ orientation); whereas, this information is lost in non-directional prep

How: Stranded/Directional prep uses dUTP in place of dTTP to label the second cDNA strand for selective degradation. This allows the first strand to be the only template left, ensuring that all of the transcripts share a common orientation

Question 17

Why Non-Stranded mRNA Library?

Answer 17

1. Simplicity: Non-stranded library prep is generally less complex and can be more straightforward to perform. It doesn’t require the additional steps needed to preserve strand information, which can simplify the workflow.
2. Cost: Because it involves fewer steps and reagents, non-stranded library prep can be less expensive than stranded library prep. This can be an important consideration for clients with budget constraints.
3. Experimental Design: Some experiments may not benefit from the additional information that stranded RNA-Seq provides. In cases where the orientation of the transcript does not impact the study’s outcome, a non-stranded will suffice. For example, if the goal is to measure overall gene expression levels rather than to study antisense transcription or overlapping genes, non-stranded RNA-Seq could be appropriate.

Why Stranded mRNA Library?
1. Strand Specificity: Stranded library prep allows researchers to determine the orientation of the RNA transcripts, which is crucial for understanding antisense transcription and enhancing transcript annotation.
2. Comprehensive Transcriptome Analysis: Stranded mRNA prep offers a clear and comprehensive analysis across the transcriptome, providing more data per assay, including
Full sequence and variant information
Higher discovery power to detect known and novel transcripts.
More accurate gene expression data
Increased alignment efficiency
Detecting features like gene fusion and allele-specific expression

Question 18

Library Prep

Answer 18

Low Input Options
Watchmaker Kit (San Jose Lab)
rRNA depletion (directional)
Input: 25ng
Human Mouse Rat only

Cost: QC (1 time High sensitive QC w/ Bioanalyzer included), rRNA Depletion Lib Prep, & Sequencing at 40M PE150 reads (NovaSeq X Plus), no analysis
< 24 Samples: $329 per sample
24-100 Samples: $309 per sample
100+ Samples: $289 per sample

Takara Kit (Outsourced to ADH)
polyA selection (non-directional)
Input: at least 10ng
Success of library prep and downstream results cannot be guaranteed

Cost: QC (1 time High sensitive QC w/ Bioanalyzer included), Takara SMART-seq V4 ultra low input RNA kit, sequencing 20M PE150 reads, no analysis
< 24 Samples: $450 per sample
24-100 Samples: $400 per sample

Question 19

Library Preparation: Globin Depletion

Answer 19

What is gmRNA?
Globin mRNA (gmRNA) is the messenger RNA that encodes globin proteins.

Globulin and gmRNA are highly abundant in whole blood, overwhelming other RNA types. Their presence can interfere with the detection of low-abundance RNA species.

gmRNA-Depletion
gmRNA is predominantly found in red blood cells. Any RNA samples derived from whole blood must undergo a globin depletion step
PBMC(s) & plasma does not typically need gmRNA-depletion

Question 20

Library Preparation: Globin Depletion

Answer 20

GLOBINclear™ Kit - Thermo Fisher Scientific: Removes predominate amount of both α and β globin mRNA via a biotinylated Capture Oligo Mix
For mRNA – paired with directional or non-directional library prep
HUMAN MOUSE RAT ONLY
Available in China Lab

TruSeq Stranded Total RNA with RiboZero Globin Depletion: Depletes samples of globin-encoding mRNA in addition to both cytoplasmic and mitochondrial rRNA using biotinylation (total RNA treatment and library preparation).
For lncRNA
HUMAN MOUSE RAT ONLY
Available in China Lab

Watchmaker Kit with rRNA and globin depletion: Uses Polaris Depletion to enhance data quality by efficiently removing rRNA and globin mRNA, improving sensitivity and coverage, particularly in low-input samples
For lncRNA
HUMAN MOUSE RAT ONLY
Available in San Jose Lab

Question 21

FFPE Tissue

Answer 21

Degradation/Fragmentation

Requires rRNA depletion

Followed by Illumina TruSeq RNA Exome Prep
This prep is targeting on human exome coding area & using rRNA depletion
Specific to humans, FFPE from other sp

Question 22

sequencing

Answer 22

choosing a sequencer
NovaSeq6000
- more expensive
- used on most historical projects
- some clients still favor it for continuity
- as less projects run on the 6000, TAT in increasing

NovaSeqX plus
- less expensive
- newest platform by illumina
- quicker
- higher through put

Question 23

calculating read depth

Answer 23

150 b.p front and back
- reads: PE150 = paired end sequencing, 150bp reads
- read pair = 2 x 150bp = 300bp read/pair