Exam 1- Lecture 6 Flashcards
What are linkers/adapters
short pieces of DNA with a known sequence
Why are linkers/adapters used
Because the end of DNA fragments are unknown after they have been sheared through sonication
What is a barcode or index sequence
sequence unique to the sample of DNA adaptor
What is multiplexing
allows for multiple samples of DNA to be analyzed at the same time
What is read depth
the number of sequences that map to a region in the genome
What is read depth used for
to ensure that a change is not an error in the sequencing process
In 454 sequencing where are beads attached to
beads via adaptors
What is present in the beads in 454 sequencing
small DNA oligonucleotides complementary to the adaptor
Once the fragments with adaptors are placed into beads what happens
one DNA fragment will attach to single bead
In Illumina sequencing, where are DNA fragments added
to the surface of a flow cell
What is on the surface of a flow cell for Illumin sequencing
DNA primers complementary to the adaptor on each of the DNA strands
For 454 sequencing, what is used to create multiple copies of the single piece of DNA
emulsion PCR
How does emulsion PCR work
emulsion of oil and water
one bead into one water droplet
water contains deoxynucleotides, primers complementary to adaptors, and Taq DNA polymerase
DNA amplified normally inside water
bead coated with identical copies of DNA
What does Illumina sequencing use to create multiple copies of DNA
bridge amplification
How does bridge amplification work
flow cells are incubated with deoxynucleotides and DNA polymerase
primers are attached to flow cell
DNA anneals to primer on surface–>forming bridge
amplified and release to form cluster of fragments
One cluster of identical strands are produced in Illumina or DNA sequencing, what happens
strands are denatured into single stranded DNAs for sequencing
How does 454 sequencing work
beads separated into picotiter plate
primer annealed to adaptor sequence
one of four deoxynucleotides and DNA polymerase floated over wells
if well has complementary base pair, nucleotide added to primer, pyrophosphate released
flash of light occurs and is recorded
How does Illumina sequencing work
After primer is annealed to adaptor sequence
all four nucleotides that are fluorescent are added to flow cell at same time
wavelength of light released at each flow cell is recorded
fluorescent dye terminator is removed and another batch is added to
What is a read
single piece of sequence information
What is contained in the well in 454 sequencing
luciferase and sulfurylase
How does Ion Torrent sequencing work
microwell filled with template DNA strand
filled with deoxyribonucleotide triphosphate
if dNTP is complementary to leading template nucleotide
dNTP is added to growing complementary strand
hydrogen ion is released
triggers ion sensor
In oxford nanopore sequencing, what is different about the flow cells
the flow cells contain nanopores embedded with electro-resistant membrane
How does oxford nanopores read DNA sequences
as DNA passes through nanopores, electrical current changes as different nucleotides pass through, resulting signal is decoded to provide specific DNA sequences
What does PacBio use as a signal for sequencing by synthesis
Light
