Sequencing Genomes Dr Hilal AY DNA sequencing Chaintermination

Are all DNA polymerases suitable for sequencing? High processivity Negligible or zero 5ʹ →

Chain-termination sequencing with Taq polymerase Figure 4. 5 Different types of primers for chain-termination

Strengths and limitations of chain-termination sequencing Figure 4. 6 read depth is needed in

NEXT-GENERATION SEQUENCING Roche 454 Illumina (Solexa) Ion torrent: Proton / PGM SOLi. D

Sanger sequencing– Next generation sequencing

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Sequencing Genomes Dr. Hilal AY

DNA sequencing

Chain-termination Sequencing

Are all DNA polymerases suitable for sequencing? High processivity Negligible or zero 5ʹ → 3ʹ exonuclease activity Negligible or zero 3ʹ → 5ʹ exonuclease activity

Chain-termination sequencing with Taq polymerase Figure 4. 5 Different types of primers for chain-termination sequencing. (A) Forward, reverse, and internal primers enable different sections of a PCR product to be sequenced. (B) A universal primer anneals to the vector DNA adjacent to the position at which new DNA is inserted. A single universal primer can therefore be used to sequence any DNA insert.

Chain-termination sequencing

Strengths and limitations of chain-termination sequencing Figure 4. 6 read depth is needed in order to identify sequencing errors. Each region of a genome must be sequenced multiple times, in order to identify errors present in individual sequence reads. In this example, the discrepancy in read 4 in the highlighted column can be ascribed to a sequencing error, the correct nucleotide at this position being C.

NEXT-GENERATION SEQUENCING Roche 454 Illumina (Solexa) Ion torrent: Proton / PGM SOLi. D

NEXT-GENERATION SEQUENCING

Roche 454 pirodizileme

Illumina/Solexa

Ion Torrent: Proton/PGM

ABI/SOLi. D

Nanopore sequencing

Sanger sequencing– Next generation sequencing

Whole genome sequencing

Single cell genome sequencing

Mitochondrial genome sequencing

Sequencing for genetic disorders