Sequencing Technology Flashcards
How big is the human haploid genome in bp?
The haploid human genome is 3.2 x10^9 bp in length
What adaptor is attached to the ends of the input DNA in illumina/next generation sequencing?
Illumina sequencing adaptors are short synthetic DNAs which contain non-complementary sequence at one end causing them to form a Y shape.
A PCR-type amplification is used to convert the Y-shaped adaptors to unique end-specific sequences (which are complementary with primers bonded to flow cell reaction surfaces)
Adaptors library molecules are pumped across flow cell surface diluted
Adaptors base pair with primers attached to flow cell
Both ends of adaptor library can interact with primers so DNA molecules form loops/bridges
PCR amplifies so there is a cluster of identical loops/bridges
Sequencer reads colour of dye attached to nucleotide
Which adaptor is attached to the input DNA of nanopore sequencing?
Nanopore adaptors are synthetic pieces of DNA which are pre-linked to lipid groups and the DNA translocations enzyme
The lipid tag allows the end of the DNA to diffuse within the lipid membrane to find a nanopore. The translocase enzyme can then be captured by the pore to begin sequencing
How does a nanopore sequencer work?
There is a pore that spans the membrane which is just big enough to allow the passage of a single strand of DNA to flow through. The pore is able to bind a motor protein called a translocase which grabs hold of dsDNA, splits it into ssDNA and threads one strand through the pore. A voltages is applied across the membrane and salt ions try to carry a measurable electric current across the membrane. Each base occludes the pore to a different extent which impedes current flow to a characteristic level.
Which adaptor is attached to the input DNA sequence of PacBio sequencing?
dsDNA template molecules are ligated to hairpin adaptors.
Hairpin adaptors are synthetic ssDNA with ends that form a region of complementary dsDNA
The hairpin adaptors template dsDNA to be opened into a large ssDNA circle
How does PacBio sequencer work?
Hairpin adaptors provide site for sequencing primers to bind. The primed template molecules are added to SMRT cell and one will engage with a DNA polymerase within a chamber.
Single DNA molecule spools through the polymerase in each chamber. As each colour-coded dNTP enters the active site, the colour of the dye is read by the imaging system. This happens transiently
What are the four problems genome sequencing technologies face?
- practical limit to the length of DNA that can be sequenced
- limit to accuracy
- a requirement for a molecular ‘handle’ by which the technology can grab the target DNA molecule
- cost
What is shotgun sequencing?
Population of DNA is broken into random fragments which can be sequenced and overlapping sequences can be pieced back together
How does illumina sequencing work?
Normal DNA synthesis is a continuous polymerisation of dNTPs (free 3’ OH group on dNTPs supports this)
The illumina DNA synthesis is discontinuous, it uses dNTPs at have been modified with a fluorescent dye in place of the normal 3’ OH group. The four dNTPs are colour coded with specific dye groups.
The illumina DNA synthesis reaction incorporates a single dye-labelled nucleotide then terminates.
Flow cell surface is washed leaving the dye incorporated at the end of the halted DNA chain
Whole flow cell slide is scanned by a laser and the colours of dye fluorescence are logged by the imaging system for each spot
Chemicals flow through flow cell removing dye groups to leave a 3’ OH
What is RNA-sequencing?
RNA-seq is a functional genomics method which can use illumina sequencing to count the number of RNA molecules transcribed from a genome. This is a technique we can use to measure levels of gene expression and map gene splicing.
Step 1: RNA is purified from a cell population, fragmented and reverse transcribed into cDNAs
Step 2: cDNAs are joined to illumina sequencer adaptors to make a library and then sequenced
Step 3: the short reads contain enough sequence information to be aligned back to a reference genome relating to the cell type in step 1
What are the different dNTPs utilised by each technology?
Sanger method - dye terminator ddTTP
Illumina method - reversible-dye terminator dTTP
PacBio method - 5’ phospho dye-labelled dTTP
