Next generation sequencing (NGS)

History of DNA/RNA sequencing

Sanger sequencing is developed in the late ’70s

nrg.2017.96-f1 1 > Source: Cieślik - 2018

RNA-seq emerged shortly after NGS from 2005

nrg.2017.96-f1 2

Source: Cieślik - 2018 Also see: rna-seqblog.com

Sanger sequencing

A.k.a: Capillary sequencing or first-generation sequencing

  • the first sequencing method still used
  • uses labeled terminating codons
  • Separation by electrophoresis / chromatography (~sep by length)
  • Readout in chromatogram

Principle of Sanger sequencing

dna-sequencing_med

The whole setup to date

Sanger-sequencing

Wikipedia

Next generation sequencing

Different NGS platforms / configurations and sequencing chemistry.

  • Illumina
  • PAC-bio
  • Ion-torrent
  • New comer: Nano-pore (very different)
Shared properties
  • massive parallel sequencing
  • spatially separated, clonally amplified DNA molecules

Illumina Sequencing

Amplification on the flow cell by bridge PCR

NGS1

Source: https://wiki.mcmaster.ca/

Sequencing of clones (local clusters) by adding coloured nucleotides

NGS2.illumina_2

Source: https://wiki.mcmaster.ca/

Sequencing by synthesis

  • Adding four fluorescently-labelled reversible terminator nucleotides, primers, and DNA polymerase to the flow cell.
  • The primer attaches to the illumina adapter of the DNA being sequenced.
  • The DNA polymerase then binds to the primer and adds the first fluorescently-labelled terminator nucleotide (base) to the new DNA strand.
  • The coloured terminator nucleotides stops it
    • All strands arrive to the same point in sequence with the
  • Microscopy (colour dots)
  • Cleave terminating residue
  • Repeat

Sequencing by synthesis - Overview

Seq.steps

Newer sequencers use only two colours to cut down the cost

Seq.colors

Single & paired-end sequencing

Paired.end.seq

Source: Devon Ryan

sc-Seq uses paired-end sequencing

  • Most scRNASeq protocols are sequenced with paired-end sequencing.
  • Barcode sequences may occur in one or both reads depending on the protocol employed.
  • Typically: one read with the CBC and UMI , other read for transcript sequence (used for mapping).
  • Reads will be mapped as if they are single-end sequenced despite actually being paired end.