Next Generation Sequencing Flashcards
Why is NGS important
-High throughput sequencing
-Lower cost over its scale
-Less time
-Parallel sequencing process
-sequence thousands of sequences at once
NGS vs NNGS
NGS- Amplified single molecule sequencing
NNGS- single molecule sequencing
It saves time and has increased efficiency
Types of library preparation
-Emulsion PCR
-‘Polony” PCR on a slide
Sequencing by synthesis
-Introduced by 454 life sciences
-specific technique is called pyrosequencing
-A fluorescently labeled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base
Library preparation steps
-Fragmenting of genomic DNA
-Ends of DNA strands repair and phosphorylated
-Tailing
-Ligate Index adaptor
-Denature and amplify for final product
Flow cell
It allows several samples to be loaded onto eight lanes for simultaneous analysis on a sequencing system
-It is a chip that contains thousands of probes with specific sequences of DNA
NGS workflow
Preparation of genomic DNA sample
-Attach DNA to surface
-Bridge amplification
-Fragments become double stranded
-Denaturing the double stranded molecules
-Complete amplification
-Determine First Base
-Image First base
-Determine second base
-Image second chemistry cycle
-sequence over multiple cycles
-align data
Preperation of genomic DNA
Randomly fragment DNA and ligate adapters to both ends of the fragments
Attach DNA to surface
Bind single-stranded fragments randomly to the inside surface of the flow cell channels
-The primers of the fragments will bind to specific oligonucleotides on the chip to organize groups of fragments
Bridge Amplification
Add unlabeled nucleotides and enzymes to innate solid-phase bridge amplification
-DNA fragments produce DNA copies on adjacent primers
Fragments become double stranded
The enzyme incorporates nucleotides to build double-stranded bridges on the solid-phase substrate
Denaturing the double stranded molecules
Denaturing leaves single stranded templates anchored to the substrate
-Only forward sequence
Complete amplification
Several million dense clusters of double-stranded DNA are generated in each of the flow cell
-Each cluster containing identical fragments
Determine First base
The first sequencing cycle begins by adding four labeled reversible terminators, primers and DNA polymerases
-The terminator ensures that only one base is added per cycle
Image First base
After laser excitation, the emitted fluorescence from each cluster is captured and the first base is identified
Determine second base
The next cycle repeats the incorporation of 4 labeled reversible terminators, primers and DNA polymerases
Image second chemistry cycle
After laser excitation, the image is captured as before and the identity of the 2nd base is recorded
Sequencing over multiple cycles
The sequencing cycles are repeated to determine the sequence of bases in a fragment, one base at a time
Align Data
The data are aligned and compared to a reference, and sequencing differences are identified
Pyrosequencing
Runs of bases produce higher peaks- multiple bases at same time
Semiconductor sequencing (Ion Torrent)
-Ion torrent started it-It is based on detection of H ions that are released during DNA polymerisation
Semiconductor sequencing workflow
Library preparation
Emulsion PCR
Semiconductor sequencing
Computer needs
512 go RAM
44 node dedicated cluster
Needs system that can handle large amounts of DATA
Sequencing of genomic DNA uses
-Whole genome sequencing
-TArgetd re-sequencing
-Epigenetic profiling
-Genomic footprinting
Sequencing of cDNA libraries uses
-RNA footprinting
Transcript one expression profiling
Transcript of mining
Limitations of NGS
-increased throughput means NGS offers shorter average read length (30-400 bp) than conventional Sanger methods (500-1kb)
-high volume of data generation (also strength) as it can range in mega bases (millions) to giga bases (billions)
