Lecture 17 & 18 Flashcards

DNA Sequence Analysis

1
Q

MPS
NGS
CS rxn
QC
DL
EM

A

Massively Parallel Sequencing
Next Gen
Cycle sequencing
Quality Control
Displacement Loop
Electron microscope

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2
Q

What are the two kinds of sequence analysis

A
  • screen for one set of known sequences (probe based)
  • Determine a novel sequence
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3
Q

Cycle Sequencing / Sanger Sequencing

A
  • similar to PCR
  • in vitro synthesis
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4
Q

What is the difference btwn Sanger and PCR ?

A
  • 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
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5
Q

What is a dye terminator reaction

A

When ddNTPs are attachted that end the sequencing

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6
Q

Seperate fragments from sangar with

A

Capillary
Smallest size = primer + 1
then primer +2 … you will get the sequence of the DNA when put together

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7
Q

What is the first thing you do for sequencing?

A

PCR

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8
Q

How long is the sequence you analyzed with Sanger sequencing?

A

the length of your PCR prod.

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9
Q

What is the advantage sequencing has with degraded DNA

A

You are able to seq smaller frag and then piece them together

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10
Q

What happens if there is a dinucleotide repeat region?

A

The electropherogram gets out of sync

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11
Q

what was used b4 fluorescence

A

radioactivityyyyyyy
Manual sequencing

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12
Q

what was a caveat to radioactivity?

A

no diff color = cant do all ddntps @ same time, so you needed to run four rxns, ea w a diff ddntp labled.

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13
Q

What does the software do for you?

A
  • applies a spectral calibration
  • apprx peak spacing
  • calls color (spacing expectation)
  • says N for low or conflicting signal
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14
Q

What does it mean when there’s an N in the sequence?

A

software cant tell what it is

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15
Q

Where is the quality of the read bad? What kind of issue is it?

A

Beginning and end
Mobility calibration issue OR unincorporated ddNTPs at the veryyyyyyyyyyyyy beginning (poor cleaning)

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16
Q

Does sequencing require a sizing standard?

A

No, relative position of peaks is used

17
Q

What is mobility?

A

a function of bp length and the diff dyes. also polymer type

18
Q

How is STR calibrated for dye mobility?

A

Allelic ladder uses the same dye as the run.

19
Q

Why do peaks become more broad at the end

A

Bc they were in the rxn for longer - random differences accumulate

20
Q

Underlying (minor) peaks

A
  • contamination
  • mixture
  • spectral calibration problem
21
Q

Homomeric stretch

A

repeat of 1 bp

22
Q

what does Homomeric stretch cause

A

strand slippage
dna molecules of slightly diff length
common in mtDNA (C)

23
Q

How do you get around the issue of Homomeric stretch’s in ur sequence?

A
  • Internal primers
  • Double rxns from same strand
  • use both stand bc before the region = clean read
24
Q

What is a strong stop?

A

peak height suddenly reduced

25
What are all the issues of sequencing electropherogram errors/issues
- Too much DNA/ PCR prod
26
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
27
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!
28
What type of DNA is usually sequenced in forensics?
mtDNA
29
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.
30
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!)
31
Why is it called D loop?
Displacement loop in EM