Next generation sequencing Flashcards
Describe the process of Roche 454 technology/sequencing.
454 technology starts by SHEARING DNA into 300-800bp fragments using RESTRICTION ENZYMES, then POLISHES the ends by removing unpaired bases.
ADAPTERS are then ANNEALED to the polished ends where the reverse strand anneals to the adapter CONTAINING BIOTIN bound to a streptavidin bead (one DNA fragment per bead). Upon binding, the bonds joining the dsDNA, break and the fragment becomes single-stranded. The forward strand anneals to the adaptor-specific primer.
OIL IS ADDED to the beads to perform emulsion PCR where each oil droplet forms its own micro-reactor. Eventually, the beads are coated with about a million IDENTICAL COPIES of the original DNA. Afterwards, the OIL IS REMOVED and each bead is placed into a well on a PICOTITER plate.
PYROSEQUENCING ENZYMES (sulfurylase, luciferase) are attached to smaller beads and added to each well. dNTPs are added one at a time, which generates PPi. Afterwards, adenosine phosphosulfate (APS) is added to work with SULFURYLASE to convert the PPi to ATP which LUCIFERASE uses to convert luciferin to OXYLUCIFERIN which generates LIGHT. The plate is coupled to a FIBRE OPTIC CHIP and a CCD camera which will record the light emitted from each well from the dNTP added, with read lengths of 0.5-1kbp. Lastly, the plate is washed to add the next dNTP in a repeating cycle.
How is Illumina sequencing performed?
Different adapters are attached to each end of the fragmented DNA, then it is bound to a slide coated with the complementary sequences for each primer for “bridge PCR” which produces a spot of amplified DNA on the slide. The spots are then visualised during sequencing from the different fluorescence emitted from each nucleotide added.
Describe Illumina Massively Parallel System (MPS) in Nextera XT.
In tagmentation, TRANSPOSASES will simultaneously fragment DNA and tag each end with a short DNA sequence. Afterwards, index primers are added to amplify and barcode fragments, limiting the number of possible PCR cycles.
The index primers contain a read (1/2) sequencing PRIMER complementary to the tag, an INDEX sequence that barcodes each sample, and a sequencing ANCHOR (P5/P7) that binds the PCR products to the flow cell.
The fragments then undergo index PCR where the index & P5/P7 sequences are INSERTED to the fragments via amplification. New bases are added one at a time, with fluorescent tags to determine which base was added and its location within the sequence. PCR is repeated 50-100x to completely sequence the DNA.
Finally, the PCR products are MULTIPLEXED and then demultiplexed computationally, to form the DNA library. This will REDUCE REAGENT COSTS and provide a QUICKER TURNAROUND TIME per sample. But REDUCES THE READ NUMBER per sample and introduces a NORMALISATION STEP to minimise variation in the read number per sample.
Why is Illumina better than Roche 454 technology?
Illumina uses fluorescent tags which will block the 3’-OH of another nucleotide, which means that another nucleotide can only be added once the fluorescent tag is removed. This allows for accurate reads. Illumina also doesn’t have the HOMOpolymer (AAAAA) issue where it cannot detect light emitted from multiple same nucleotides.
What are the different shearing methods for DNA?
DNA is mechanically sheared via sonication or centrifugal force (G-tube). And, enzymatically sheared with transposase, restriction enzymes, or nicking enzymes.
What are the benefits/applications of Paired-end indexed sequencing such as in PacBio?
Paired-end indexed sequencing is required for discovering GENOME VARIATIONS. It enables better COVERAGE UNIFORMITY by anchoring repetitive sequences with unique paired reads. It can detect INSERTIONs & DELETIONs by searching for reads with UNUSUAL DISTANCEs between pairs.
What are the benefits of PacBio?
Short waiting time, produces long reads, no amplification required, allows for direct observation.
How does PacBio with Single Molecule, Real-Time (SMRT) sequencing work?
In SMRT sequencing, DNA is fragmented into smaller sequences and the ends are LIGATED with hairpin loop adaptors to each fragment end to create a circular template with a primer binding site and initiation sequence. The circular template allows the polymerase to continue onto the reverse strand to produce long reads.
This prepped sample is then added to the SMRT cell where each fragment is immobilised into tiny wells called zero-mode waveguides (ZMWs) with a polymerase affixed at its bottom. ZMW represents the lowest available volume for light detection capable of detecting a single nucleotide for accurate reads. During sequencing, the polymerase will add dNTPs each with different fluorescent tags linked at the terminal phosphate so that when a dNTP is detained by the polymerase the phosphate bonds break and fluorescence is emitted to enable real-time sequencing.
There are 2 sequencing modes. Circular consensus sequencing (CCS) mode is for small inserts that generate multiple passes on each molecule to produce accurate reads. Long sequencing (LS) mode is for large inserts that generate one pass on each molecule for the longest possible reads.
POLYMERASE KINETICS will measure the duration between two successive base insertions from the intervals between fluorescent pulses which is called interpulse durations (IPD). IPDs are altered in MODIFIED BASES due to METHYLATION which affects gene expression or ALTERED GENE FUNCTION which causes malignant cellular transformations. Hence, polymerase kinetics can detect any modified base.
How does Oxford Nanopore-Sequencing work?
DNA is fragmented during sample preparation then a helicase will unzip the fragments into a single-stranded DNA and feed it through a nanopore in an electrically resistant membrane (a nucleotide at a time) by complexing with a processing enzyme. When a potential is applied, each base blocks the ion flow to a different degree which identifies them as they pass through the nanopore. The longest read length is >1Mbp.
