NGS Flashcards
Generally how does NGS sequencing work?
Couple synthesis and detection
Many sequencing reactions run in parallel
Sequencing by synthesis
- Sequential addition of dNTPs (Ion Torrect, 454)
- Simultaneous addition of all dNTPs but only one nucleotide added in each round of sequencing
What is 454 sequencing?
Launched by the 454 corporation and aquired by Roche, it was shut down in 2013
454 using pyrosequencing
Based on the detection of light whenever a nucleotide is incorporated.
What were the issues with 454 sequencing?
Homopolymers - difficult to determine correct homopolymer length, resulting in deletion/insertion of polymers
Amplification errors - up to 15% of the resulting sequence was a result of in vitro amplification
Loss of synchronous light emissions - some wells fall out of step due to left over nucelotides or incomplete extension/
How does the Ion Torrent sequencing work?
Ion torrent exploits the release of a H+ ion with the addition of each nulceotide. Like the 454 method, it uses fragmentation adding beads to emulsion PCR. Beads are then added to wells on a slide. Each nucelotide is added sequentially and the change in pH is measured to determine how many nucleootides added.
What are the strengths and weaknesses of ion torrent sequencing?
Strengths:
- Relatively cheap
- Fast, limiting step is the sequential addition of dNTPs
Weaknesses:
- Homopolymer regions, difficult to exactly determine length
What are the four steps in illumina sequencing?
- Sample prep
- Cluster generation
- Sequencing by synthesis
- Base Calling
What happens during the illumia sample prep?
- DNA is fragmented so the majority of DNA is 100-300bp
- End damage is repaired and A added to the 3’ end to improve ligation in the next step.
- Illumina adapters are added
- Size selection is carried out (200-400bp). Fragments that are too long will create overlapping clusters
- PCR enrichment is carried out with the P5 and P7 primers acting as amplification elements. The number of PCR cycles needs to be optimised to give sufficient material without introducing PCR bias.
- The result of Sample prep is double stranded DNA with oligo sequencing for indexing, sequencing primer binding and attachment to a flowcell.
What happens during the illumina cluster generation?
Cluster generation takes place on a flow cell. A flow cell is divided into lanes, each lane is coated with oligos complementary to sequences on the ends of our fragments. Individual fragments are used as seeds for generating clusters by bridge amplification.
In bridge amplification, DNA is denatured to a single strand and added to a flow cell where it binds to complementary primer sequence. A complementary strand is generated and the two strands are seperated and the original washed away. How we have a sequence which is attached to the surface of the slide.
DNA polymerase and dNTPs are added and a complementary strand synthesised.
This is repeated until a cluster of forward and reverse sequences are all physically joined at the slide at the 5’ end. The reverse strands are then removed
What happens during the illumina sequencing by synthesis?
- Sequencing is carried out using coloured fluorophore attached nucleotides.
- A,C,T and G are added at the same time
- Only one nt can be added in each round of sequencing
- Therefore all sequences will be the same length
- Homopolymer regions should be less of a problem
- A laser excited the fluorophore and light is emitted
- An optic scanner reads the signal
After the first round of sequencing is completed the read product is washed away. Another round of bridge amplification is carried out and it is cleaved at the primer site so that the reverse strand is now the only one remaining.
Illumina used four different dyes for four bases. Four different images are created using different filters for the four different emissions to determine which base has been added at each cluster.
In the NextSeq it uses two dyes
What is used to enable multiple samples to be run on an illumina platform?
Multiplexing uses short unique sequences (barcodes) inserted into primers to identify each sample.
Following sequencing they are demultiplexed into different files.
The number of samples which can be multiplexed on a run depends on the sequencer throughput, the coverage needed and the size of the regions
What stage(s) of sequencing causes duplicates to arise?
PCR duplicates - PCR amplification of fragments with adapters
Cluster duplicates - Bridge PCR amplifying the single molecule on each bread or each clone of the flow cell.
Why does the sequencing quality decrease during a run?
Reduction in sequencing quality is caused by phasing errors. Phasing occurs when a wrong nucleotide is added causing the following sequence to be out of phase. Once out of phase, the sequencing cannot get back in phase and as more and more sequences become out of phase it means the base cannot be called as confidently and therefore the sequencing quality decreases.
What is SMRT sequencing and which company produces it?
SMRT (single molecular real time) sequencing is produced by PacBio. As it is a real time analysis, there are no pauses between read steps. Sequencing data is produced at the speed that the DNA polymerase adds the nucleotide.
This produced much longer reads than Illumina/454/Ion Torrent and has a lower throughput than Illumina.
SMRT sequencing uses ZMW (zero mode waveguide) structures. These are an array of tiny wells in aluminium. DNA polymerase is immobilised at the bottom of these wells. Modified nucleotides are added to create a cimplementary strand. Each dNTP has a fluorescent lable which fluoresces as it is added. After addition the fluorescent label is cleaved.
Sigle pass sequence reads in SMRT sequencing are more error prone (~11%) hoever errors are randomly distributed. There is a consensus accuracy of >99.999%
How do longer reads improve sequencing analysis?
Longer reads improve mapping and enable identification of rearrangements such as CNVs and structural rearrangements.
What is nanopore sequencing?
Unlike other sequencing methodologies, nanopore does not sequence by synthesis and instead directly sequences DNA molecules.
Nanopore sequencing is fast and produces incredibly long reads. The longest read to date is >200,000nt. It can also directly sequence RNA molecules and can detect modifications such as methylation.
It is real time data analysis, you get the results as the DNA strand goes through the pores.
The long reads of the nanopore make it ideal for carrying out de novo sequencing of genomes.
There has been a series of improvements improving the speed and accuracy. The accuracy is now at 94% and increasing.
