next generation sequencing

Question 1

why do we sequence the whole genome

Answer 1

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

Question 2

how is DNA extended

Answer 2

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

Question 3

what are the 3 main stages of PCR

Answer 3

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

Question 4

what are the main steps are Sanger sequencing

Answer 4

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

Question 5

how many base pers can Sanger do per sequence

Answer 5

up to 700

Question 6

what is bases are included in Sanger sequencing

Answer 6

nucleotides and dideoyxnucleotides- labelled terminators

Question 7

How does Sanger extend and work out sequencing

Answer 7

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

Question 8

what anaylses the results of sanger sequencing

Answer 8

capillary electrophoresis and a laser reader

Question 9

what is the disadvantage of sanger

Answer 9

low output redepth of 1

Question 10

how is the addition of a base detected in 454 technology

Answer 10

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

Question 11

what is the main issue with 454 technology

Answer 11

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%

Question 12

Which method is optimised for small genomes

Answer 12

illumina

Question 13

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

Answer 13

enzymatic and because you use small genomes

Question 14

what do transposons do

Answer 14

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

Question 15

how long does illumination take

Answer 15

in up to 2 days can get around 10 billion reads

Question 16

how can you sample pool

Answer 16

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

Question 17

what do the p5 and p7 so in illumina

Answer 17

allow the DNA to bind to the flow cell

Question 18

what are the advantages of sample pooling?

Answer 18

-reduces reagent cost
-quicker time around time per sample

Question 19

what are the disadvantages of sample pooling

Answer 19

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

Question 20

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

Answer 20

required for gene variation and is used in illumina

Question 21

what is the advantages of paired end indexed sequencing

Answer 21

-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.

Question 22

how does paired end indexed sequencing work

Answer 22

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

Question 23

How does 454 detect the light

Answer 23

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

Question 24

What is the size of reads from Pacbio

Answer 24

Much bigger at 10kb to 20kb

Question 25

What are advantages of pacbio

Answer 25

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

Question 26

What type of dna shearing does pac bio

Answer 26

Mechanical: sonofication or g tubules

Question 27

What is enzymatic shearing used for

Answer 27

Small dna sequences- if too big would cut too small

Question 28

How does sonificstion work

Answer 28

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

Question 29

What advantage does g tubule have over sonification and disadvantage

Answer 29

Cheaper but has lower throughput

Question 30

How do g tubules work

Answer 30

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

Question 31

What is needed for sample prep after dna shearing in Pacbio

Answer 31

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

Question 32

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

Answer 32

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

Question 33

How does Pacbios way of adding fluoroflores differ to illumina

Answer 33

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

Question 34

How does pacbio detect fluorescent signals

Answer 34

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

Question 35

How does pacbio detect flourcence

Answer 35

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

Question 36

What are the advantages of pac bio

Answer 36

Short waiting time Long reads Direct observations No amplification required

Question 37

What is the advantages of oxo nanowire

Answer 37

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

Question 38

What is the disadvantage of nanopore

Answer 38

It’s accuracy is in question 90-99%

Question 39

How does nanopore work

Answer 39

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

Question 40

What accuracy and error is a factor phred score of 20

Answer 40

99% and 1/100

Question 41

What accuracy and error rate is a phred score of 30

Answer 41

1/1000 and 99.9%

Question 42

What could cause a poor distribution per base quality

Answer 42

General degradation of quality over duration of long runs

Question 43

What do the different colours mean in the phred scores

Answer 43

Colder mean higher quality Warmer mean Lower quality

Question 44

What causes low quality scores and what do u do

Answer 44

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

Question 45

How to tel if GC content is bad

Answer 45

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

Question 46

What might over representation indicate

Answer 46

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

Question 47

Why are there gaps in de novo assembly

Answer 47

Because of repetive sequences

Question 48

How is reference mapping done

Answer 48

Against a reference genome

Question 49

What does reference mapping do

Answer 49

Allows you to identify single nucleotide polymorphisms, insertions and deletions

Question 50

What programs do reference mapping

Answer 50

Bwa Bowtie

Question 51

What program allows manual inspection of sequences and mapped reads

Answer 51

Artemis

Question 52

What is the difference between synomous snp and non synonyms snp

Answer 52

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

Question 53

What if the difference between missense and non sense snps

Answer 53

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

Question 54

What is trams

Answer 54

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