genome analysis - sequencing techniques Flashcards
Which sequencing techniques requires amplification?
Illumina
IonTorrent
Which sequencing techniques are short read techniques?
Illumina
IonTorrent
Which sequencing techniques are for long read sequencing?
Pacific Bioscience
Oxford Nanopore
Which techniques are SMRT techniques?
PacBio
ONT
How does Illumina sequencing work?
What is the result?
It uses sequencing by synthesis.
Adapters are attached to the fragments and they are attached to a flowcell. On the flowcell, the fragments are amplified into clusters using bridge amplification.
Labeled nucleotides are then added and fluorescent signals indicate when a nucleotide has been incorporated.
High quality, high throughput reads of 100-150 bp with very low indel error rate, mismatches are more common. Illumina reads have very high quality in the beginning of the reads so trimming of the reads is usually necessary.
How does IonTorrent sequencing work? What is the result?
Each fragment attaches to a bead where they are amplified. Each bead is placed in a well on the chip and the well is flooded with one nucleotide at a time.
When a nucleotide is incorporated hydrogen ions are released and there is a pH change in the well. This change is converted to voltage which then can give us a basecall.
Produces reads of 200, 400 or 600 bp depending on the chip.
Homopolymers are challenging to sequence so the indel error rate is comparatively high and remains even with high coverage, all errors occur almost equally.
How does PacBio sequencing work? What is the result?
SMRT sequencing which means that the technique uses the natural replication process.
Adapters are attached to double stranded fragment creating a circular template. Primers and polymerase is added and the library is placed in wells where the polymerase works. When a nucleotide is added correctly, light is emitted.
Can produce either:
- circular consensus reads (HiFi) which have very high accuracy (99.8%) 10-20kbp.
- continuous long read - get the longest read possible (~50kbp).
SMRT have high error rates (~15%) as the signal to noise ratio from single DNA molecules is not high. This is best resolved using HiFi reads. The error rate is higher for indels than mismatches.
How does oxford nano pore sequencing work? What is the result?
Single molecule sequencing.
Sequence until satisfied.
Can be used in field.
Fragment is sequenced by threading it through a microscopic pore in a membrane. Bases are identified by the way they are blocking the current of ions moving from one side of the membrane to the other.
Produces long reads, longest ~4Mb with error rate of 1-5%.
Has issues with homopolymers but not a severe as IonTorrent.
What are Phred scores?
Quality metrics for sequencing reads.
It tells you probability of the base being correct. Maximum shred score is ~40.
What is the quality of PacBio reads?
High error rate (15-30%) due to signal-to-noise ratio is very small for single molecules.
We have a random error distribution because the reads are of different length. This together with the fact that the reads are long, means that we can still find the overlaps even though the error rate is so high.
