Molecular bio Wk 7

What are the multiple methods for DNA sequencing?

-Sanger DNA sequencing uses -advanced DNA sequencing methods

What are Sanger DNA sequencing uses?

-chain termination to determine bases in sequence -requires that DNA synthesis is terminated to determine sequence

What are advanced DNA sequencing methods?

-mostly use continuous incorporation of bases into growing DNA chain to determine sequence -do not use chain terminators (ddNTPs) -sequencing by synthesis -use a variety of methods to read DNA sequence -massively parallel methods (next generation sequencing (NGS))

What is Pyrosequencing (basis)?

-principle of 'sequencing by synthesis' -based on DNA synthesis and dynamics of nucleotide incorporation into new DNA strand -nucleotide incorporation causes release of pyrophosphate -pyrophosphate initiates a chain of reaction that ultimately leads to light emission

What is NGS (Next Generation Sequencing)?

-massively parallel, high-throughput DNA sequencing methods -millions of DNA fragments are sequenced simultaneously, making it fast and cost-effective

What are short-read examples?

illumina, ion torrent

What is short read sequencing?

high accuracy, lower cost, good for small genomes or exomes (reads of ~100-300 bp)

What are examples of long-read?

pacbio, oxford nanopore

What is long-read sequencing?

better for structural variation, repetitive regions, de novo assembly (reads up to 10000+ bp)

What is the basis of Illumina Sequencing?

-sequencing by synthesis -DNA fragments are attached to a flow cell and amplified (clonal clusters) -reversible dye-labelled nucleotides are added one at a time -a camera captures fluorescence after each base is added

What is the basis of Ion Torrent Sequencing?

-sequencing by synthesis, but NO LIGHT -detects hydrogen ions released when DNA polymerase adds a base -pH change is detected by a semiconductor chip

What are the different methods of NGS target enrichment?

-hybridisation-based capture -amplicon-based sequencing

What is hybridisation-based capture?

-biotin labelled probes complementary to targets of interest -hybridise with fragmented DNA -pulled down by streptavidin-coated magnetic beads

What is Amplicon-based?

pools of PCR primers used to PCR amplify target regions in huge multiplex PCRS

What is NGS Validation?

variations identified often need to be confirmed by other genotyping methods (e.g. Sanger sequencing)

What are the NGS Limitations?

-high startup costs -expensive to sequence WGS, WES -computationally intensive