DNA sequencing

Sanger

uses dideoxynucleoside triphosphates

sequencing reaction is like PCR with a single primer

dideoxynucleoside triphosphates

lacks 3’OH group

terminates DNA-chain elongation because 5’H cannot act as nucleophile in phosphodiester bond formation

Sanger advantages

• template used as plasmid or PCR product

• high-confidence in determination of sequence

• 700-800nts of continuous sequence

• Easy to analyze resulting data

Sanger disadvantages

• expensive

• need to know enough sequences to design a primer

Sanger sequencing reaction with PCR and one primer step 1

have template of unknown sequence

Sanger sequencing reaction with PCR and one primer step 2

add DNA polymerase, 4dNTPS, 4ddNTPS

Sanger sequencing reaction with PCR and one primer step 3

dye-labeled segments of DNA copied from template with unknown sequence

Record

Sanger sequencing reaction with PCR and one primer step 4

dye-labeled segments subjected to gel electrophoresis

Sanger sequencing reaction with PCR and one primer step 5

computer0generated results after bands migrate past detector

Next generation: illumina, nanopore

sequence many distinct template molecules in parallel in miniature

Illumina advantages

• sequence whole genome cheaply

• sequence whole plasmid cheaply

• Nothing specific needs to be known about sequence in advance no specific primers

• Identify SNPs

Illumina limitations

• sometimes shorter reads (depends on platform)

• Higher error rates

• Not cost effective for small scale project

• analysis may require computational power and expertise

Common uses of next-generation sequencing

• sequence plasmids

• sequence new genome (genomes of species whose genomes have not been sequenced)

• Sequence genomes from individuals or strains or cell lines or single cells to identify different mutations

• measure gene expression

Steps of Illumina 1

take DNA genome and cut into fragments

Steps of Illumina 2

Adaptors added to fragments to construct library

Steps of Illumina 3

Sequencing

Steps of Illumina 4

Data analysis

Illumina sequence uses

modified nucleotide derivates

1. added blocked 3’end flourescently labeled nt

2. remove labels and blocked groups; wash; add blocked labeled nucleotides

Illumina sequencing

reads aligned to reference genome

Oxford nanopore sequencing

may be used instead of or in addition to illumina (genome) or sanger plasmid)

sequencing can be aligned to

reference or assembled de novo

de novo (new) better when expecting big difference from existing reference

Oxford nanopore sequencing advantages

• No PCR during library construction; sequencing DNA directly

• reads can be long (10kbs) allow assembly without need for reference sequence

Oxford nanopore sequencing drawback

high error rates