Sequencing Technologies Flashcards
Which generation of sequencing is Sanger?
1st gen
What are the characteristics of first generation sequenced reads?
Fairly long, individual reads (roughly 1kb).
The reads are high quality
If you wanted to produce a targeted sequence, for example a PCR product, which sequencing technology would you be likely to use?
1st generation, for example Sanger dideoxy sequencing
What generation of sequencing is Illumina?
2nd (next) generation sequencing
What are the characteristics of next generation sequencing?
Massively parallel sequencing
Short high quality reads (300-500bp)
What are four instances where you would want to use next generation sequencing?
Functional genomics (eg RNAseq or ChIPseq)
Draft genome sequences (cannot resolve repeat sequences so cannot be entire genome)
Re-sequencing a genome against a reference
Metagenomics - sequencing of complex populations of organisms
What are the two types of 3rd generation sequencing?
Oxford Nanopore and PacBio
What type of reads do 3rd gen sequencing machines produce?
Single molecule sequence, v long (roughly 10kb)
What is third generation sequencing good for?
Finishing genomes (with no gaps) and identifying base modifications (epigenetics)
What is a limitation to third generation sequencing?
Individual reads can have quite a high error rate
discuss in brief how illumina sequencing works
Genomic DNA is fragmented by sonication or enzymatically
Size select fragments of roughly 500bp
Add linkers of a known sequence (at both ends of fragment) which will allow the fragment to be amplified
Linkers bind to primers on flow cell
In situ PCR using bridge amplification to generate clusters
Sequence
What does each flow cell cluster correspond to in Illumina sequencing?
Each cluster is derived from a single initial molecule, and each cluster corresponds to a different read.
What are the limitations surrounding the yield of reads in standard bridge amplification techniques?
If clusters are too densely packed, you cannot resolve individual reads.
The density of a cluster determines the total yield of reads.
If the density is too high, clusters cannot be resolved and this limits the maximum number of reads you can resolve from an input
What are the benefits of using a patterned flow cell?
Patterned flow cells use tiny nanowells on the surface of the flow cell rather than normal flat flow cell.
Clusters build up in the wells so there is no cluster overlap
How are the clusters formed in patterned flow cell pure?
Cluster build up in each well is fast enough that it prevents secondary molecules falling in the well
