Sequencing and transcriptomics
🧬

Daniel Fürth
Assistant professor
SciLifeLab/Uppsala University

🔗 furthlab.xyz
@furthlab

📚🙇🏽‍♀️🙇🏻‍♂️📖 Learning goals

Course Syllabus: 🔗3MG022

Knowledge and understanding

“Explain current techniques for research, diagnostics and treatment of genetic diseases including cancer”

Competence and skills

“independently analyse, process and formulate relevant scientific questions…”

Judgement and approach

“…ability to critically evaluate and appraise scientific research…”

📚🙇🏽‍♀️🙇🏻‍♂️📖 Learning goals

Topics covered today:

• 📜 History: from Sanger sequencing to single-cell methods
• 🔎 Transcriptomic analysis
• 🧬 Different types of RNAs and their functions

📜 Sanger sequencing ➡️ single-cell methods

Separation chemistry

Electrophoretic machine as designed by Tiselius

Separation chemistry

Polyacrylamide gel electrophoresis (PAGE)

• Length
• Charge
• Confirmation

Molecules can be run in their “native state”
preserving higher-order structure.

Or a chemical denaturant can be added to
remove structure (urea for 🧬).

Native vs non-native PAGE.

Pore size controled by % of acrylamide.

📏 Resolution:
100-1000 nt down to single-nucleotide.

Fredrick Sanger

• One of the few who received two Noble Prizes.
  • 💉1958

    • “structure of proteins, especially that of insulin”

  • 🧬1980

    • “determination of base sequences in nucleic acids”

🇬🇧 Medical Research Council
Laboratory of Molecular Biology

Left MRC Laboratory of Molecular Biology, 1962. Right Left to right: Hugh Huxley, 🏅John Kendrew (’62), 🏅Max Perutz (’62), 🏅Francis Crick (’62), 🏅Fred Sanger (’58,’80) and
🏅Sydney Brenner (2002).

🛠🧰 Restriction enzymes 🧬

🇺🇸 Restriction and modification enzymes 🇨🇭

🏅Noble Prize: 🇺🇸 Hamilton O. Smith, 🇺🇸 Daniel Nathans, and 🇨🇭 Werner Arber.

Left to right: 🇨🇭Grete Kellenberger, 🇨🇭 Werner Arber, 🇨🇭 Daisy Dussoix

Structure of a Ribonucleic Acid

Structure of a Ribonucleic Acid

RNase A (Bovine pancreas ribonuclease):

• Only active on RNA.
• Very sturdy, survives boiling etc 🔥.
• Cleaves 3’-end of unpaired C and U residues (pyrimidine).

RNase T1 (Taka-Diastase ribonuclease T1):

• Single-stranded (ssDNA or RNA) specific exonuclease
• Catalyzes the removal of nucleotides from linear single-stranded DNA or RNA in the 3’ to 5’ direction

Nuclease Protection Assay (NPA)

A proto sequencing method.

Two-dimension partition sequencing

A proto sequencing method.

• First seperate on isoelectric point (pI)
  • pH @ molecule has no net electric charge.
• Then seperate on molecular weight.

Two-dimension partition sequencing

Two-dimension partition sequencing

dNTP incorporation has base specificity

The birth of the “-”-approach of Sanger.

First sequence of a single gene

• Bacteriophage MS2
• Circular ssDNA genome.

Sanger’s Plus/Minus sequencing

Sanger’s Plus/Minus sequencing

Dideoxynucleotides (ddNTP) are incorporated

Polymerases can incorporate ddNTPs.

Leading to a single base extension followed by termination

No 3’hydroxyl group (3’OH).

🏆 Chain-termination method 🏅

“Sanger sequencing”

Second Noble Prize

Maxam & Gilbert sequencing

• Radioactive 5’labeling

• Chemical cleavage

• PAGE

The birth of biotech

California. Genentech and recombinant technologies were already creating value. Flow cytometer and Beckman Coulter commercial machines.

⛰ Cal. Tech. 👥 Leroy Hood and Mike Hunkapiller 🏢 Applied Bioscience Inc. (ABI)

Sanger sequencing

Fluorescently labeled nucleotides.

Capillary gel electrophoresis

Sanger sequencing

Base calling: assigning specific nucleotide bases to raw signals generated during sequencing.

Human Genome Project

Next-generation sequencing

All about how to massively multiplex sequencing.

• Pyrosequencing

• Sequencing by ligation

• Reversible dye-terminators

Solid-phase substrate

• Bridge amplification

• Emulsion PCR

• DNA nanoballs

Primer extension

• Sequencing by synthesis
  (SBS)

• Sequencing by ligation
  (SBL)

Pyrosequencing

• Principle: DNA polymerase incorporates nucleotides, releasing pyrophosphate (PPi).

• Signal: PPi is converted to light via sulfurylase and luciferase, where the light intensity indicates the number of incorporated bases.

• Analysis: Light signals are used to determine the DNA sequence in real time.

• Question: How can individual bases (A, T, C, G) be discriminated?

Polony sequencing

Solexa/Illumina sequencing

• Solid support: PCR bridge amplification
• Primer extension: Reversible dye-terminators (patent 444 expired last year!)

Solexa/Illumina sequencing

Reversible dye-terminators

Bridge amplification

Bridge amplification - Flow cell

• Flow cell: A microscope slide where oligonucleotides have been functionalized with a linker so that they are covalently attached to the glass.

Bridge amplification - Illumina adapters

• P5 adapter: common that the first read (R1) starts form this end.
• P7 adapter: common that the second read (R2) as well as the multiplex index (what sample) reads from this end (i7 = index P7).

Bridge amplification - sekvenseringsbibliotek

• PCR used to flank our DNA library with P5 and P7 adapter sequence.

• The library cannot be directly placed on the flow cell because it is double-stranded DNA (dsDNA) and therefore cannot bind to the flow cell oligos.

Bridge amplification - denaturering och laddning

• The sequencing library is diluted to very low concentrations together with NaOH (sodium hydroxide).

• NaOH is strongly basic, which denatures dsDNA into ssDNA.

Bridge amplification - denaturering och laddning

• The sequencing library is diluted to very low concentrations together with NaOH (sodium hydroxide).

• NaOH is strongly basic, which denatures dsDNA into ssDNA.

• The library is pumped onto the flow cell, and the pH is adjusted down, causing the ssDNA library to bind to the P5 and P7 oligos via hybridization.

Bridge amplification - PCR

• ssDNA annealed to flowcell

Bridge amplification - PCR

• Polymerase extension from P5/P7 adapter.

Bridge amplification - PCR

• dsDNA

Bridge amplification - PCR

• dsDNA

Bridge amplification - PCR

• Increase temperature och wash away non-covalent ssDNA.

Bridge amplification - PCR

• Decerase temperature
• Hybdrize to complementary adapter at the flow cell.

Bridge amplification - PCR

• Decerase temperature
• Hybdrize to complementary adapter at the flow cell.
• Polymerase copies the covalent ssDNA.

Bridge amplification - PCR

• Decerase temperature
• Hybdrize to complementary adapter at the flow cell.
• Polymerase copies the covalent ssDNA.
• dsDNA

Bridge amplification - PCR

• Decerase temperature
• Hybdrize to complementary adapter at the flow cell.
• Polymerase copies the covalent ssDNA.
• dsDNA
• Increase temperature and start the PCR cycle again.

Democratization of sequencing

RNA sequencing

• Is actual DNA sequencing.

• But the RNA is converted to cDNA by reverse transcriptase.

RNA sequencing

Single-cell RNA sequencing

🔎 Transcriptomic analysis

Primary, secondary, tertiary

Gene body and why to sequence

• Alternative splicing

• Intron retention, pre-mRNA

• Alterative polyadenylation (3’UTR exon)

Strand-specificity

• Allele-specific expression

🧬 Different types of RNAs and their functions

🧬 Different types of RNAs and their functions

🧬 Different types of RNAs and their functions