Technologies for massive parallel sequencing
🧬

Daniel Fürth
Assistant professor
SciLifeLab/Uppsala University

🔗 furthlab.xyz
@furthlab

Technologies for massive parallel sequencing

📚🙇🏽‍♀️🙇🏻‍♂️📖 Lärandemål

Kurplan: 🔗3MG010

Innehåll:

• Kromosomstruktur, normal och avvikande kromosomuppsättning.
• Monogen och multifaktoriell nedärvning.
• Metoder för gen-identifiering vid enkla och komplexa egenskaper.
• Populationsgenetik, riskberäkning i familjer.
• Diagnostik inom klinisk genetik, screening av nyfödda samt bärare.
• Det humana genomets sammansättning och genetisk variation.
• Genomevolution, genetiska modellorganismer och komparativ genomik.
• 🧬 Metoder att analysera hela genoms struktur och funktion, storskalig analys av DNA-sekvens och epigenetisk variation, samt mätning av transkriptions- och proteinnivåer.
• Metoder för att koppla en gen till en sjukdom.
• Mekanismer för reglering av geners uttryck.
• Användandet av genetiska markörer inom forensisk medicin. Etiska principer, processer och deklarationer.
• Farmakogenetik och cancergenetik.

📚🙇🏽‍♀️🙇🏻‍♂️📖 Lärandemål

Saker som vi ska gå igenom idag:

• 📜 Historiskt perspektiv:
  • från Sanger-sekvensering till Next-Generation Sequencing (NGS)
  • 🔴🟢🟣🔵 Sanger-sekvensering genom kapillärelektrofores
• NGS solid-phase substrate:
  • 🪨 Emulsion PCR
  • 🪨 Bridge amplification
• NGS primer extension:
  • 🧪 Sequencing by Synthesis (SBS)
  • 🧪 Pyrosequencing
  • 🧪 Reversible-dye terminators (Illumina)
  • 🧪 Sequencing by Ligation (SBL)
    
• 🧬 Long read sequencing
  • PacBio
  • NanoPore

📜 Sanger sequencing ➡️ Next-Generation Sequencing (NGS)

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.

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

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

Sanger sequencing

Capillary gel electrophoresis

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)

Human Genome Project

Technological landscape

• Read length
• Throughput

Read length

Throughput

Alignment

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

Emulsion PCR/Polony sequencing

Emulsion PCR

Solexa/Illumina sequencing

• Solid support: PCR bridge amplification
• Primer extension: Reversible dye-terminators (patent 444 about to expire!)

Solexa/Illumina sequencing

Solexa/Illumina sequencing

Solexa/Illumina sequencing

Sequencing by Ligation (SBL)

Democratization of sequencing

Paired-end and mate pair

DNA library preparation

🧬 Long read sequencing

Single Molecule, Real-Time (SMRT) sequencing

• PacBio
  • SMRTbell Template Prep
  • Zero-Mode Waveguides

NanoPore sequencing

• OxfordNanopore

Unbiased in situ sequencing

In situ sequencing