Next generation sequencing (NGS)

History of DNA/RNA sequencing

Sanger sequencing is developed in the late ’70s

> Source: Cieślik - 2018

RNA-seq emerged shortly after NGS from 2005

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

The whole setup to date

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

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

Sequencing of clones (local clusters) by adding coloured nucleotides

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

Newer sequencers use only two colours to cut down the cost

Single & paired-end sequencing

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.