Lecture 17 & 18 Flashcards
DNA Sequence Analysis
MPS
NGS
CS rxn
QC
DL
EM
Massively Parallel Sequencing
Next Gen
Cycle sequencing
Quality Control
Displacement Loop
Electron microscope
What are the two kinds of sequence analysis
- screen for one set of known sequences (probe based)
- Determine a novel sequence
Cycle Sequencing / Sanger Sequencing
- similar to PCR
- in vitro synthesis
What is the difference btwn Sanger and PCR ?
- only 1 primer
- not exponential copies, arithmetic increase
- PCR product is the template, so you start w/ a lot
- Some dNTPs are ddNTPs DIdeoxy = missing 3’ hydroxyl preventing the addition of another nucleotide
- The ddNTPs are labled w a dye that indicates what base it is = dye terminator reaction
- then capillary
What is a dye terminator reaction
When ddNTPs are attachted that end the sequencing
Seperate fragments from sangar with
Capillary
Smallest size = primer + 1
then primer +2 … you will get the sequence of the DNA when put together
What is the first thing you do for sequencing?
PCR
How long is the sequence you analyzed with Sanger sequencing?
the length of your PCR prod.
What is the advantage sequencing has with degraded DNA
You are able to seq smaller frag and then piece them together
What happens if there is a dinucleotide repeat region?
The electropherogram gets out of sync
what was used b4 fluorescence
radioactivityyyyyyy
Manual sequencing
what was a caveat to radioactivity?
no diff color = cant do all ddntps @ same time, so you needed to run four rxns, ea w a diff ddntp labled.
What does the software do for you?
- applies a spectral calibration
- apprx peak spacing
- calls color (spacing expectation)
- says N for low or conflicting signal
What does it mean when there’s an N in the sequence?
software cant tell what it is
Where is the quality of the read bad? What kind of issue is it?
Beginning and end
Mobility calibration issue OR unincorporated ddNTPs at the veryyyyyyyyyyyyy beginning (poor cleaning)
Does sequencing require a sizing standard?
No, relative position of peaks is used
What is mobility?
a function of bp length and the diff dyes. also polymer type
How is STR calibrated for dye mobility?
Allelic ladder uses the same dye as the run.
Why do peaks become more broad at the end
Bc they were in the rxn for longer - random differences accumulate
Underlying (minor) peaks
- contamination
- mixture
- spectral calibration problem
Homomeric stretch
repeat of 1 bp
what does Homomeric stretch cause
strand slippage
dna molecules of slightly diff length
common in mtDNA (C)
How do you get around the issue of Homomeric stretch’s in ur sequence?
- Internal primers
- Double rxns from same strand
- use both stand bc before the region = clean read
What is a strong stop?
peak height suddenly reduced
What are all the issues of sequencing electropherogram errors/issues
- Too much DNA/ PCR prod
What is the issue with Too much PCR Prod?
difficult to distinguish adj peak of same color
unavoidable when seq short amplicon
PCR is efficient when you sequence a short length… affecting ss
What is relationship btwn Phred score and prob base was called wrong?
Bigger Pred score = smaller chance it was called wrong
Its the lil bar graphs above the sequence!
What type of DNA is usually sequenced in forensics?
mtDNA
How can you tell if a sequence contains a protein coding gene? Contains a pseudogene?
if there is a sequence of codons
If there is a stop codon in the middle.
What is the single non-coding hypervariable region in mtDNA
Vertebrates: D loop / Ctrl region
Invertebrates: A+T rich region
~1 kb
accumulation of polymorphic sites (ID!)
Why is it called D loop?
Displacement loop
in EM
