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analytical exam 2 > next generation sequencing > Flashcards

next generation sequencing Flashcards

1
Q

why do we sequence the whole genome

A

-biodiveristy and speciation
-diversity within a species
-biology of organism

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2
Q

how is DNA extended

A

through 3’0H group of the pentose sugar by 5’ phosphate of free nucleotide. Phosphodiester bond formed and diphosphate released.

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3
Q

what are the 3 main stages of PCR

A

-denaturation
-anneal primer
-extend new strand by incorporating dNTPs

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4
Q

what are the main steps are Sanger sequencing

A

dna fragenatation, clone into vectors, transform bacteria, grow and isolate vector DNA. Sequence librbay and assemble contiguous fragments

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5
Q

how many base pers can Sanger do per sequence

A

up to 700

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6
Q

what is bases are included in Sanger sequencing

A

nucleotides and dideoyxnucleotides- labelled terminators

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7
Q

How does Sanger extend and work out sequencing

A

Bind primer to known part of DNA sequence allowing DNA pol to bind extend sequence until get to terminator. This happens over and over again with the terminator at different positions

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8
Q

what anaylses the results of sanger sequencing

A

capillary electrophoresis and a laser reader

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9
Q

what is the disadvantage of sanger

A

low output redepth of 1

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10
Q

how is the addition of a base detected in 454 technology

A

if a nucleotide is added phosphate is released. In the presence of ASP and sulfurylase, ATP is released which can be used to make light and oxyluciferin

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11
Q

what is the main issue with 454 technology

A

if there is a homopolymer it is hard to detect light flashes from one nucleotide vs many nucleotides. If 5As vs 6As is only 20%

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12
Q

Which method is optimised for small genomes

A

illumina

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13
Q

what type of DNA shearing would you use for illumina and why

A

enzymatic and because you use small genomes

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14
Q

what do transposons do

A

they cut the DNA fragmenting it and then add adapter to the DNA

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15
Q

how long does illumination take

A

in up to 2 days can get around 10 billion reads

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16
Q

how can you sample pool

A

can add an index sequence which is 8-12 bp taht allows you to pool mulitiple fragments together.

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17
Q

what do the p5 and p7 so in illumina

A

allow the DNA to bind to the flow cell

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18
Q

what are the advantages of sample pooling?

A

-reduces reagent cost
-quicker time around time per sample

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19
Q

what are the disadvantages of sample pooling

A

Reduced read number per sample
-introduces normalisation step to minimise variation in read number per sample

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20
Q

what is paired end indexed sequencing needed for and which technology uses it

A

required for gene variation and is used in illumina

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21
Q

what is the advantages of paired end indexed sequencing

A

-enables better coverage uniformity by allowing high receptive sequncinhg to be anchored by unique pair read
-insertion and deletion of event can be detected by searching for reads that have an unusual distance between their pairs.

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22
Q

how does paired end indexed sequencing work

A

one ion the olgios on the floor cell is complementary to the

23
Q

How does 454 detect the light

A

Plate is coupled with a fibre optic chip. A CCD camera record the light

24
Q

What is the size of reads from Pacbio

A

Much bigger at 10kb to 20kb

25
Q

What are advantages of pacbio

A

Bigger read
Can span repeat regions to determine sequences and close up genomics

26
Q

What type of dna shearing does pac bio

A

Mechanical: sonofication or g tubules

27
Q

What is enzymatic shearing used for

A

Small dna sequences- if too big would cut too small

28
Q

How does sonificstion work

A

Fire ultra focused sound waves at different wavelengths at your dna which breaks it to the size you want

29
Q

What advantage does g tubule have over sonification and disadvantage

A

Cheaper but has lower throughput

30
Q

How do g tubules work

A

They fire DNA through a fine mesh in a centrifuge which breaks down DNA. How fast u spin changed the size of your DNA

31
Q

What is needed for sample prep after dna shearing in Pacbio

A

Smart bell formation
-fragment
- repair ends
-ligate adapters
- purify dna
- sequencing

32
Q

What are the two different sequencing modes in Pacbio and which is better

A

LS- long sequence reads
CCS- Hugh quality sequencing reads
CCS looks at shorter fragments but does multiple copies so can fix if error

33
Q

How does Pacbios way of adding fluoroflores differ to illumina

A

Uses triphospahte linked fluorophores to reduce steric hinderance which allows DNA pol to move quicker

34
Q

How does pacbio detect fluorescent signals

A

Zero mode wavelengths hold fluorescent signal and can detect base incorporated despite back ground of other nucleotides

35
Q

How does pacbio detect flourcence

A

Through zero model wave guides where a single c ocular dna and dna pop is. A laser excited the floourorphore and fluorescence is emitted and recorded

36
Q

What are the advantages of pac bio

A

Short waiting time
Long reads
Direct observations
No amplification required

37
Q

What is the advantages of oxo nanowire

A

Can sequence extra long reads
No amplification needed
Can use pcr but don’t have to
Can look at DNA and RNA

38
Q

What is the disadvantage of nanopore

A

It’s accuracy is in question
90-99%

39
Q

How does nanopore work

A

A current passes through the nanopore
When the bases come through they disrupt the ion flow and each base disrupts differently giving differnt reads

40
Q

What accuracy and error is a factor phred score of 20

A

99% and 1/100

41
Q

What accuracy and error rate is a phred score of 30

A

1/1000 and 99.9%

42
Q

What could cause a poor distribution per base quality

A

General degradation of quality over duration of long runs

43
Q

What do the different colours mean in the phred scores

A

Colder mean higher quality
Warmer mean Lower quality

44
Q

What causes low quality scores and what do u do

A

Imaging problems or technical problems
Need to remove it from down stream so isn’t patty of analysis

45
Q

How to tel if GC content is bad

A

There will be a sharp peak where there should be a smooth curve which suggests there could be confinement

46
Q

What might over representation indicate

A

Over resperenatation of a sequence indicates taht the libabry might be contaminated

47
Q

Why are there gaps in de novo assembly

A

Because of repetive sequences

48
Q

How is reference mapping done

A

Against a reference genome

49
Q

What does reference mapping do

A

Allows you to identify single nucleotide polymorphisms, insertions and deletions

50
Q

What programs do reference mapping

A

Bwa
Bowtie

51
Q

What program allows manual inspection of sequences and mapped reads

A

Artemis

52
Q

What is the difference between synomous snp and non synonyms snp

A

Synmous - no change to aa
Non synmous- change to aa

53
Q

What if the difference between missense and non sense snps

A

Missense- differnt as coded
Nonsense- results in premature stop codon

54
Q

What is trams

A

It’s a rapid annotation toon for annoattaion of microbial snp
Searches for multiple nucleotide polymorphisms

analytical exam 2 (6 decks)

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