Technologies for massive parallel sequencing

Daniel Fürth
Assistant professor
SciLifeLab/Uppsala University


Technologies for massive parallel sequencing

📚🙇🏽‍♀️🙇🏻‍♂️📖 Lärandemål

Kurplan: 🔗3MG010


  • 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.

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



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

  • Sequencing by ligation


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