Lecture 17 & 18

Question 1

MPS
NGS
CS rxn
QC
DL
EM

Answer 1

Massively Parallel Sequencing
Next Gen
?
Quality Control
Displacement Loop
?

Question 2

What are the two kinds of sequence analysis

Answer 2

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

Question 3

Cycle Sequencing / Sanger Sequencing

Answer 3

• similar to PCR
• in vitro synthesis
• differences from PCR:

Question 4

What is the difference btwn Sanger and PCR ?

Answer 4

• 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

Question 5

What is a dye terminator reaction

Answer 5

When ddNTPs that end

Question 6

Seperate fragments from sangar with

Answer 6

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

Question 7

What is the first thing you do for sequencing?

Answer 7

PCR

Question 8

How long is the sequence you analyzed with Sanger sequencing?

Answer 8

the length of your PCR prod.

Question 9

What is the advantage sequencing has with degraded DNA

Answer 9

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

Question 10

What happens if there is a dinucleotide repeat region?

Answer 10

The electropherogram gets out of sync

Question 11

what was used b4 fluorescence

Answer 11

radioactivityyyyyyy

Question 12

what was a caveat to radioactivity?

Answer 12

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

Question 13

What does the software do for you?

Answer 13

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

Question 14

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

Answer 14

software cant tell what it is

Question 15

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

Answer 15

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

Question 16

Does sequencing require a sizing standard?

Answer 16

No, relative position of peaks is used

Question 17

What is mobility?

Answer 17

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

Question 18

How is STR calibrated for dye mobility?

Answer 18

Allelic ladder uses the same dye as the run.

Question 19

Why do peaks become more broad at the end

Answer 19

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

Question 20

Underlying (minor) peaks

Answer 20

• contamination
• mixture
• spectral calibration problem

Question 21

Homomeric stretch

Answer 21

repeat of 1 bp

Question 22

what does Homomeric stretch cause

Answer 22

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

Question 23

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

Answer 23

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

Question 24

What is a strong stop?

Answer 24

peak height suddenly reduced

Question 25

What are all the issues of sequencing electropherogram errors/issues

Answer 25

• Too much DNA/ PCR prod

Question 26

What is the issue with Too much PCR Prod?

Answer 26

difficult to distinguish adj peak of same color
unavoidable when seq short amplicon
PCR is efficient when you sequence a short length… affecting ss

Question 27

What is relationship btwn Phred score and prob base was called wrong?

Answer 27

Bigger Pred score = smaller chance it was called wrong

Its the lil bar graphs above the sequence!

Question 28

What type of DNA is usually sequenced in forensics?

Answer 28

mtDNA

Question 29

How can you tell if a sequence contains a protein coding gene? Contains a pseudogene?

Answer 29

if there is a sequence of codons
If there is a stop codon in the middle.

Question 30

What is the single non-coding hypervariable region in mtDNA

Answer 30

Vertebrates: D loop / Ctrl region
Invertebrates: A+T rich region
~1 kb
accumulation of polymorphic sites (ID!)

Question 31

Why is it called D loop?

Answer 31

Displacement loop
in EM