Sequencing technologies Flashcards
Explain the general procedure of Illumina
- Create library molecules consisting of the relevant sequences by fragmenting, polishing and ligating adapters
- PCR amplify
- Sequence in parallel
Explain emulsion PCR
Primers of the same kind are fixated on a bead in a solution of water. The template associates with the primer, and the amplification takes place. The template then dissociates. The beads are then linked to a glass slide.
Explain bridge amplification
A DNA template (one per cluster) is embedded on the surface together with primers. It folds over to meet the other primer (bridge formation). Then the synthesis takes place. The original strand is washed away, and the newly synthesized bends over and a new, “original” strand is made. The two strands separate (F+R) and the process is repeated to form clusters.
What is the main bias using sequence technologies that employ PCR?
You only find what you are looking for (primer selection)
Why should Illumina not be used for amplicon sequencing?
Because many of the same bases being incorporated at the same time gives a big flash that “hides” any other colours
What is index hopping? How to avoid it?
When the library is assigned to a wrong index in one of the ends. Dual indexing and not having free adapters in sample prevents it.
454: How does it work?
What is main weakness/strength?
Memo: Flooding Fragment DNA (1kb), add adapters and do emulsion PCR with adaptor-specific primers. Then flood the slide with one NTP at a time and observe is one or more is added by emission of fluorescent light signals.
Cannot separate long homopolymer stretches, but can sequence long reads
How does the Illumina sequencing work downstream of bridge amplification?
What is the main weakness?
Reverse strands are washed off and primers are attached to the forward strands to which fluorescent nts are added. The incorporation of a bases gives a light signal. The the indexes are sequenced, and the reverse strand is sequenced.
The DNA polymerase accuracy declines throughout the reaction
IonTorrent
Based on conductivity - release of H+ ions when base is incorporated. Flooding with a different nt. Emulsion PCR.
BGIseq
DNA is fragmented, adapters are ligated to it, and the strand circularizes. A DNA pol synthesizes the complementary strand and it keeps synthesizing by zipping up previous base pairs (rolling circle) –> DNA nano ball. The DNB is loaded on a slide, and sequence data is determined base by base (by flooding labelled probes) and high-resolution image is constructed after every incorporation.
Gives short reads
Oxford Nanopore
Many small pores (no shit Sherlock). A strand of DNA passes through the pore giving rise to different changes in the current. Has high error rate but long reads.
PacBio
Immobilized DNA polymerase in the bottom of the well, incorporates nts that are labelled on the phosphate part.
Can give long reads which minimizes errors. Can be used for epigenetics.
PacBio
Immobilized DNA polymerase in the bottom of the well, incorporates nts that are labelled on the phosphate part.
Can give long reads which minimizes errors. Can be used for epigenetics.
NextSeq/NovaSeq: Pros and cons
2 dyes, faster and cheaper
Higher error rate, esp. with G stretches (no dye)
How does 10x Genomics work:
Library preparation and sequencing technology
single cell sequencing method that uses beads and unique bar-codes to indetify reads.
Bar-codes are unique from bead to bead and another unique identifier UMI for scRNA seq that makes every single reads unique within the bead
10x genomics uses illumina techonology for sequencing
