week 2: mRNA seq

Question 1

RNA vs. DNA

Answer 1

Molecular differences

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)

RNA
- AGUC
- OH on sugar
- single stranded, and often but not always, linear in shape

DNA
- AGTC
- H on sugar
- two antiparallel, complementary strands form a double helix

Question 2

central dogma

Answer 2

DNA, RNA, mRNA, protein

transcription: DNA to RNA

polyadenylation: RNA to mRNA

translation: mRNA to protein

Question 3

mRNA

Answer 3

– 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 4

mRNA vs. “Other” RNA

Answer 4

Other types of RNA are important for many applications as well, however, a main focus within the genomics is on actively expressed transcripts (result of transcription; cell makes RNA out of DNA), that is why mRNA is specifically so important

Other types:
- tRNA, lncRNA, ncRNA, rRNA, miRNA, snRNA, snoRNA

Question 5

TLDR:

Answer 5

“Gene Expression” study = mRNA Seq

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

Question 6

QC steps

Answer 6

total RNA

(sample QC)

library construction

(library QC)

sequencing

(data QC)

Bioinformatics analysis

Question 7

QC & Library Prep
- Starting Material

Answer 7

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)
- we encourage clients to perform their own RNA extraction is possible

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 8

Sample QC
- QC Method = Agilent 2100 or AATI fragment analyzer

Answer 8

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 9

Sample QC
RNA Sample Requirements

Answer 9

Client Facing Sample Requirements

chart of requirements - look in ppt :)

Question 10

RNA Sample Requirements
- Qubit

Answer 10

Quantification of RNA
- Unit: ng/µL

Not used as part of Novogene internal QC, but is a common tool used by 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 11

RNA Sample Requirements
- Nanodrop

Answer 11

Quantification & Qualification of RNA
Units: ng/µL & Absorbance Units (A)
Not used as part of Novogene internal QC, but is a common tool used by 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 12

RNA Sample Requirements
- Agilent 2100 Bioanalyzer / AATI Fragment analyzer / Tapestation

Answer 12

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 13

Library Prep Options

Answer 13

bug chart look at ppt :)

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

so it is named after what it eliminates basically

Question 15

Library Preparation: PolyA Selection vs rRNA Depletion

Answer 15

rRNA-Depletion uses

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

Note: rRNA depletion preps are always directional. polyA selection preps can either be directional or non-directional

Question 17

Library Prep: Directional vs Non-Directional Prep

Answer 17

chart look at ppt :)

Question 18

Library Prep: Directional vs Non-Directional Prep
- Why Non-Stranded mRNA Library? - so why non-directional?

Answer 18

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.

Question 19

Why Stranded mRNA Library?

Answer 19

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.

Comprehensive Transcriptome Analysis: Stranded mRNA prep offers a clear and comprehensive analysis across the transcriptome, providing more data per assay, including
1. Full sequence and variant information
2. Higher discovery power to detect known and novel transcripts.
3. More accurate gene expression data
4. Increased alignment efficiency
5. Detecting features like gene fusion and allele-specific expression

Question 20

Library Prep
- Low Input Options

Answer 20

Watchmaker Kit (San Jose Lab)
- rRNA depletion (directional - better for looking at antisense studies and studies that need direction lol and for understanding the transcriptome). rRNA eliminates rRNA and is used if the client wants ncRNA
- 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, less complex so less expensive and is good if client’s study does not require directionality)
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

Prioritize Wachmaker kit for all low input samples (prob. because we need to outsource it and also that it is cheaper), UNLESS
- BSL concern
- Previous batch of samples was processed at ADH
- Non HMR species

Question 21

Library Preparation: Globin Depletion

Answer 21

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

peripheral blood mononuclear cells (PBMCs)

Question 22

Library Preparation: Globin Depletion

Answer 22

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 23

Library Preparation
- FFPE Tissue

Answer 23

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