Week 4

Question 1

__________ are a result of sequence similarity. They introduce errors to the measured expression levels

Answer 1

Microarrays: cross hybridization

Question 2

how can cross hybridization be controlled for

Answer 2

• good probe design
• sequence alignment software
• stringent washing step
• modeling cross hybridization effects and reducing this noise source

Question 3

What are is the process of RNA sequencing:

Answer 3

• isolate all mRNA
• convert to cDNA using reverse transcriptase
• sequence the cDNA/ amplified DNA
• map sequence (reads) into the genome

Question 4

RNA sequencing is a ___________ because the final number of reads corresponding to RNA’s from one gene color is proportional to the original number of genes RNA molecules in the cell

Answer 4

random sampling process

Question 5

If the read is aligned uniquely to gene A, it is very likely this read was generated from mRNA of ____________

Answer 5

gene A

Question 6

After aligning all the reads to the genome, assume gene A gets 1000 reads ad gene B has 100 reads (aligned to its exons)> These numbers can be used to compare expression levels of the two genes. What does this mean

Answer 6

It means that Gene A is more expressed than gene B though both are the same length

Question 7

RNA sequencing is ______________ whereas miccroarrays are _____________

Answer 7

• direct sequencing of cDNA

- hybridization to pre designed probes

Question 8

microarray chips are getting _______________

Answer 8

bigger, better, and cheaper

Question 9

Net generation sequencing: speed increases and cost _______________

Answer 9

drops exponentially

Question 10

______________ is one sequencing run that exerted 1 billion reads

Answer 10

illumina nexseq 2000

Question 11

which end of the adaptor is immobilized?

Answer 11

The 5’ end because when you want to make a new strand of DNA you go from the 3’ end to the 5’ end

Question 12

___________ occur when new fragments being over to star another round of PCR

Answer 12

Bridge amplification

Question 13

What end of the DNA is attached to the flow cell surface

Answer 13

both have the 5’ end immobilized

Question 14

sequencing by synthesis of the first base:

Answer 14

1. fourescenttely labeled
2. Has a terminator
3. Terminator binding is reversible

Question 15

First sequencing cycle begins by adding _____________

Answer 15

fluorescent dNTPs, primers, and DNA polymerases

Question 16

Why not sequence before bridge amplification?

Answer 16

IF you only had one copy then that one fluorescent molecule is not that sensitive and could not be defected

Question 17

After many cycles of next generation sequencing

Answer 17

the rate off sequencing error increases

Question 18

___________ is similar to adapter lighted cDNA fragment library as illumina

Answer 18

solid sequencing

Question 19

Instead of generating clusters in the flow cell, we use small __________ to amplify the fragments

Answer 19

beads

Question 20

What are the major components of ABI solid sequencing

Answer 20

• the substrates
• Di base probes
• 5 universal seq primers

Question 21

________ are floiurcently labeled DNA probes

Answer 21

Di base probes

Question 22

How many different probes can their be

Answer 22

(16*4^3) = 1024

Question 23

How many different dye base probes

Answer 23

16

Question 24

Sequencing by ligation

Answer 24

1. Prime and ligate
2. image
3. Cap unextended strands
4. Cleave off Fluor
5. repeat steps 1-4 to extend sequence
6. primer reset
7. repeat steps 1-5 with a new primer

Question 25

Universal sew primer (n-1) is shifted too the left by 1 base relative to primer ______________

Answer 25

n | *there are 3 other primers: n-2, n-3, n-4

Question 26

___________ is output data from the sequencer are a series off color which represent dinucleotides

Answer 26

double interrogation

Question 27

Theoretical decoding of color space data: _________ if we know the sequence of the first base

Answer 27

theoretical color data spacing

Question 28

Why do we go through the trouble of color spacing out intermediates

Answer 28

By converting to color space we are preventing future errors because of the nature of the color space, if one color is off the entire sequence is off. Thus converting to color spaces help us control for small errors

Question 29

How do you know which one is a single nucleotide mutation and which one is a sequencing error

Answer 29

The odds of getting two sequencing errors in a row is small thus when there is only one different color base it is likely a sequencing error

Question 30

What are two consequences of 3rd generation sequencing

Answer 30

- long reads - no amplification required - low coverage - high error

Question 31

what are some consequences of 2nd generation sequencing

Answer 31

- high coverage - amplification step - errors tend to be systematic - great for quantification

Question 32

What is an example of 3rd generation sequencing?

Answer 32

Pacific bioscience | *single molecule real time San sequencing (SMRT)

Question 33

In Smart sequencing errors occur ______________

Answer 33

randomly, so gif we sequence the same thing multiple times we can catch error

Question 34

________ in 3rd generation DNA sequencing allows us to sequence the same thing multiple times

Answer 34

circular library

Question 35

What is the main reason for sequencing error in 3rd generation sequencing

Answer 35

Interpherance caused by background noise

Question 36

What are the pros and cons of pacific bio science

Answer 36

pro: long read, single molecule con: high error, expensive

Question 37

___________ determines the sequence of DNA fragments by passing DNA through a protein (or other) pore in a membrane

Answer 37

nanopore sequencing

Question 38

Oxford became the first company to provide a

Answer 38

commercially available sequencer to the community

Question 39

Nonopore:

Answer 39

pro: extremely long sequences of up to a single molecule, portable cons: high error rates