8: DNA Sequencing Flashcards
What is DNA sequencing?
A method to determine the exact order of nucleotide bases in a DNA fragment.
Chain termination sequencing/
Sanger sequencing/
Dideoxy sequencing
Synthesizing partial copies of the target DNA that vary in length by one nt and then separating them by size through a capillary electrophoresis tube. Smallest fragment travels longest.
Fluorescent laser excites fluorophore at 3’ end. The wavelength/color of the light is measured and recorded as G, A, T, or C. Smallest fragment first.
Reaction occur via cycles in a thermocycler.
Only used one primer (not 2 like PCR) => one strand is copied.
What are the components needed for synthesizing the different length fragments in sequencing reactions?
Template DNA
DNA pol: genetically engineered with thermal stability higher processivity and less exonuclease activity.
Buffers and ions
Sequencing primer complementary to the target DNA.
Pool of deoxyribonucleoside triphosphates (dNTPs) for the 4 bases.
Mixture of dideoxynucleoside triphosphates (ddNTPs) for the 4 bases, which are missing the -OH gr. on 3’ C => halt of DNA synthesis if incorporated (termination).
Each ddNTP has different fluorophore.
What is next generation sequencing, NGS?
Uses massively parallel methods where millions of sequencing rx. occur and are recorded simultaneously
What are the main steps of next generation sequencing?
- Genomic DNA (gDNA) isolation
- NGS library construction
- gDNA fragmentation into small ds pieces of about equal size (tagmentation or ultrasonic disruption)
- Modifying the fragments so they are compatible with the sequencing platform (adding adapters with known seq to blunt ends=>primers can anneal) - Partitioning the library fragments into separate locations on a solid surface and create clusters of identical copies.
- Sequence each cluster
- Analyze the data
What is Illumina sequencing by synthesis?
Partitioning of the individual gDNA fragments on a flow cell, and then creating a cluster of identical DNAs for sequencing using bridge amplification.
How is the library preparation of illumina sequencing?
Ultrasonic disruption:
Sound waves breaks bonds holding DNA backbone together => fragments
The two strands must be equal/blunt ended, and 5’ ends must be phosphorylated for the adapter to anneal.
Single A overhang can help adapter to anneal.
Tagmentation:
Two adapters bound to transposase mix with gDNA => cut, and tagging with adapters at the ends.
Adapters:
- Sequence that is complementary to the oligonucleotides that are bound to the flow cell.
- Index seq that is unique to the genomic fragments from one sample.
- Seq that is complementary to the sequencing primer.
PCR amplification step:
Can add index sequence. Important when gDNA from different individuals are sequenced together (multiplexing).
Illumina sequencing:
How is the library partitioned and clustered generated?
Uses flow cell:
Glass slide with microfluidic channels on the surface that allow sequencing reagents such as DNA polymerase, dNTPs, buffers and sequencing primers to flow over the surface.
Lower surface has oligonucleotides (PCR primers) attached to the glass that provide anchor points for the gDNA fragments to attach.
After attaching, clusters are generated via PCR; bridge amplification.
Illumina sequencing:
How is gDNA sequenced by synthesis?
Uses reversible dye terminators to record the nt as a G, A, C, or T.
The dye termination is reversible, and after removal, DNA pol can add another nt.
The final seq for each cluster is called a read. There are four reads for each cluster.
Illumina sequencing:
How is NGS data analyzed?
Reference genome vs de novo sequencing
- Combine the reads from each cluster.
Two of the reads decoded the index seq on both adapters. - Alignment into continuous seq information by comparison to a previously sequenced (reference) genome, or by comparing different reads looking for overlapping seq.
Variant:
Nt seq found in the gDNA sample that differs from the reference genome, which can include single nt polymorphisms (SNPs), insertions or deletions (indels), or chromosomal aberrations.
De novo sequencing:
- Decoding the nt order for an organism without a reference genome.
- Goal is to put together as many reads into one continuous length of sequence information (contig).
- May need to make consensus seq: idealized base seq consisting of the bases most often found at each position.
Read depth:
The number of reads that cover a location. Larger r.d. makes it easier to determine a mistake.
What is ion torrent sequencing?
Partitions individual gDNA by first attaching the fragments to a bead and then uses emulsion PCR to create the thousands of copies.
How is the library preparation of Ion torrent sequencing?
Library preparation similar to Illumina; small DNA fragments with adapters added to each end.
The fragments are attached to microbeads coated with a complementary oligonucleotide to the fragment’s adapter seq. One fragment per bead.
Emulsion PCR:
Beads separated in drops of liquid suspended in an oil solution.
PCR reagents are water soluble and partition into the water droplets.
After multiple PCR cycles, each bead has thousands of gDNA fragments.
Ion torrent sequencing:
How is gDNA sequenced by synthesis?
Each bead is partitioned into separate microwells on a semi-conductor chip.
Have pH meters that measure the release of protons (H+) during phosphodiester bond formation.
pH change is converted into a voltage change and recorded as a 1 or 0 by the computer.
1 = nt added
0 = nt not added
Faster than Illumina.
Weakness: if gDNA has multiple identical bases in a row. Hard to detect the voltage differences => bases must be added separately.
What is targeted sequencing?
Isolating a series of genes or regions of interest from a whole gDNA sample before sequencing using NGS technologies.
Often only sequence the (protein encoding) exons: whole exome sequencing
Methods of creating a targeted DNA sequencing library:
- Multiplexed PCR rx. to amplify the regions of the genome of interest. The PCR amplicons are attached to adapters, sequenced, and compared to reference genome as in other NGS rx.
- Biotinylated oligonucleotide probes that have seq complementary to the chosen targeted genes or exons.
Isolation, fragmentation, add adapters.
Fragment mixed and annealed to biotinylated probes. Mixed with magnetic beads coated with streptavidin => only probed gDNA bind streptavidin.
Heating => detachment of gDNA from beads and probes => analysis of gDNA.
What is nanopore sequencing?
Third-generation sequencing technique.
Determining the order of bases of a ss of DNA as it passes through a small pore or channel in a membrane.
A detector records the characteristics of the ss DNA as it passes through the pore. The normal ionic current is reduced as the DNA passes, depending on the base seq (G>C>T>A)
