RNA Seq

Question 1

method of choice to study gene expression and identify novel RNA species

Answer 1

RNA seq

Question 2

Since RNA seq is done using instruments that sequence DNA molecules what step must take place

Answer 2

cDNA library prep from RNA

Question 3

Most common application of RNAseq

Answer 3

sequencing of polyadenylated RNA

Question 4

oligo-dT priming based methods can exhibit

Answer 4

3’ bias
results in sequencing reads enriched for the 3’ portion

Question 5

Enzyme that converts RNA into DNA

Answer 5

Reverse transcriptase

Question 6

Most abundant type of RNA

Answer 6

ribosomal

Question 7

why do we not want ribosomal RNA

Answer 7

usually of little interest to the study as they make up ribosomes that make proteins, not genetic material

Question 8

Methods used to remove rRNA

Answer 8

poly A selection
rRNA depletion

Question 9

preferred method to remove rRNA

Answer 9

poly A selection

Question 10

why would you use the method of rRNA depletion

Answer 10

if interested in noncoding RNAs

Question 11

Which of the two methods to delete rRNA are more expensive

Answer 11

rRNA

Question 12

What is the next step after removing rRNA

Answer 12

fragmentation to a certain sample size range

Question 13

why does lack of strand specificity make it difficult to identify the antisense and novel RNA species

Answer 13

less unique sequence makes it harder to piece together

Question 14

Creating a library for RNA

Answer 14

incorporate a dUTP into the second strand of cDNA

Question 15

almost all multi-exon genes display

Answer 15

alternative splicing which plays a role in regulation of cellular processes

Question 16

Gene fusion

Answer 16

can place two noncontinuous genomic regions together in a single transcript

Question 17

solution to unravel the complexity of alternatively splicing and gene fusion isoforms is to

Answer 17

sequence each transcript from beginning to end

Question 18

what is important for gene regulation

Answer 18

what
when
where
how much

Question 19

types of changes in reading frame

Answer 19

silent-replace AA but similar enough not to matter
synonymous- changes sequence but not AA
nonsynonymous-changes AA

Question 20

The power of RNA seq lies in the fact that the twin aspects of

Answer 20

discovery and quantification can be combined in a single throughput sequencing array

Question 21

Crucial prerequisite for a successful RNA seq study is that

Answer 21

the data generated have the ability to answer the biological questions of interest
experimental design

Question 22

SE

Answer 22

want to know how much

Question 23

PE

Answer 23

want to know which

Question 24

how many protein coding genes in mammalian genome

Answer 24

20,000

Question 25

total number of distinct molecules and how much we sequence depends on

Answer 25

library complexity

Question 26

two types of replicates

Answer 26

technical-accuracy of technique
biological-what we are interested in, true variation

Question 27

Why are three replicates the minimum

Answer 27

3 is just enough to compare data for statistical reasons
always better to have more

Question 28

analyzing methods

Answer 28

genome mapping
transcriptome mapping
reference free assembly

Question 29

why would too many short sequences cause a problem

Answer 29

short reads give quantity not quality

Question 30

transcript expression is

Answer 30

how much
what level the gene is expressed

Question 31

Normalizing to the size of the gene

Answer 31

important step for normalizing the data

Question 32

in the absence of biological replication

Answer 32

no population inference can be made

Question 33

results of RNA seq are affected by

Answer 33

parameter