Genomes and Sequencing

Question 1

What do you do before you can start to sequence?

Answer 1

Extract DNA and fragment it

Question 2

What is shotgun sequencing?

Answer 2

Splitting up DNA into fragments randomly to be sequenced

Question 3

How is shotgun sequencing achieved?

Answer 3

Sonifaication

Question 4

What is the best sequencing method to use in reality?

Answer 4

Illumina

Question 5

What type of PCR goes along with Illumina?

Answer 5

Bridge PCR

Question 6

Why is Illumina the best to use?

Answer 6

454 (pyrosequencing) has a homopolymer problem i.e. can’t distinguish between strings of same nucleotide and can’t get volume
Ion torrent is mostly for specialised sequencing
Sanger is old
Nanopore not consistent, high error rate

Question 7

Why shouldn’t you discuss all of them?

Answer 7

Because that would make it too fragmented and it wouldn’t work in real life

Question 8

What is an amplicon?

Answer 8

A PCR product

Question 9

What should your target coverage be and why?

Answer 9

30x because above that you probably can’t improve the error rate anymore

Question 10

What should your length be?

Answer 10

At least 15 - 20 base pairs

Question 11

How does Illumina work?

Answer 11

After PCR, fluorescently label all bases with different colour (reversible terminator bases) and synthesise complementary strand of DNA to already acquired strand. Bases compete to form a regular second strand of DNA by matching up with base pair, other 3 are washed away and the camera can detect the fluro colour so allowing us to work out the original base.

Question 12

Outline your methodology for sequencing a genome

Answer 12

DNA extracted
Fragmented by sonification shotgun sequencing
Adaptors added to both ends of a fragment
Adaptors attach to inside of flow cell
Bridge PCR performed
After amplification, nucleotides labelled with own fluorescent colour
Bases compete to form regular complementary strand, opposite nucleotide binds and the other 3 are washed away
Observe colour under light to work out original base
Modified dNTP inhibits extension at 3’ end so only 1 base added at a time
Resulting contigs (overlapping bits of DNA) are scaffolded by filling in the gaps and linking

Question 13

Describe the assembly process

Answer 13

Ordering sequenced DNA fragments into genome using paired end reads
Contigs are consensus read of fragments so they are grouped to create a contig
Contigs lined up and joined using paired end data resulting in scaffolds

Question 14

What is meant by coverage?

Answer 14

The number of times a section of the genome is represented in the sequenced fragment

Question 15

What is meant by identity?

Answer 15

The percentage of the reads that agree with the same nucleotide

Question 16

What are identity and coverage used for?

Answer 16

To discount errors and differentiate between errors and heterozygosity. 50% or close indicates heterozygosity

Question 17

What is bin size?

Answer 17

When boundaries are set to distinguish between statistical variation and error

Question 18

What is genomics?

Answer 18

The study of all the genes of a cell or tissue at the DNA, mRNA or protein level

Question 19

What is comparitive genomics?

Answer 19

The study of the relationship of genome structure and function across different biological species or strains

Question 20

What is functional genomics?

Answer 20

The study of gene and protein functions and interactions utilising the data produced by genome projects

Question 21

What are the 2 methods of gene duplication?

Answer 21

Nonhomologous recombination and retrotransposition

Question 22

What is unequal crossing over?

Answer 22

Two non homologous DNA double helices align and undergo recombination which is greatly facilitated if the two strands already contain repeat units

Question 23

What are the 4 levels of duplication?

Answer 23

Exons duplicate/shuffle to change size of function of genes
Entire genes duplicate to make multigene families
Multigene families duplicate to produce gene superfamilies
Genomes duplicate to double number of copies of every gene and gene family

Question 24

What is a multigene family?

Answer 24

A set of genes descended by duplication and diversification from one ancestral gene
Can be tandem or dispersed

Question 25

What are pseudogenes?

Answer 25

The decomposing remnants of either failed duplication events, transposition genes or disabled genes Unnecessary copies of genes

Question 26

What are tandem arrays?

Answer 26

The same sequence repeated over and over

Question 27

What is the crossover fixation model?

Answer 27

Duplication of a gene is likely to result in an immediate relaxation of selection on the new members of the gene family but there are instances where all of the duplicated genes retain the same sequence and function e.g. rRNA genes

Question 28

What are Hox genes?

Answer 28

A family of genes that specify the anterior/posterior axis and segment identity of metazoan organisms during early embryonic development which produce regulatory transcription factors

Question 29

How did Hox genes arise?

Answer 29

Gene duplication

Question 30

What is the immunoglobulin superfamily?

Answer 30

A large and functionally diverse group of proteins that share a common structural feature, the immunoglobulin fold The fold can interact with other Ig folds to form dimeric molecules, display an enormous diversity of recognition sites and resist proteolysis in the blood

Question 31

What is site-specific recombination?

Answer 31

Rag1 and Rag2 enzymes catalyse reactions that result in a single V, D and J segment. They are combined and the others are excised. Different combinations lead to increased diversity

Question 32

What are transposable elements?

Answer 32

DNA segments that transpose themselves

Question 33

Describe what happens when transposition occurs to an already replicated recipient site

Answer 33

Transposition occurs to the opposite strand leaving a vacant donor site Replication is completed Vacant donor strand remains on one strand No net increase in number of Ac elements

Question 34

Describe what happens when transposition occurs to an unreplicated recipient site

Answer 34

Transposition occurs to parental strand before replication fork Completion of replication Net increase in number of Ac elements

Question 35

Name the 4 classes of transposons

Answer 35

LINEs SINEs Long Terminal Repeat retrotransposons DNA transposons

Question 36

What is simple transposition?

Answer 36

Cut and paste | Bacteria and eukarya

Question 37

What is replicative transposition?

Answer 37

Copy and paste Uncommon Bacteria

Question 38

What is retrotransposition?

Answer 38

Common in eukarya

Question 39

Where are direct repeats?

Answer 39

Found within the host DNA

Question 40

Where are inverted repeats?

Answer 40

At the ends of most transposable elements

Question 41

Describe the structure of a transposon

Answer 41

Inverted repeats at the ends | Encode transposase to recognise inverted repeats

Question 42

How does transposase work?

Answer 42

Cuts at the borders between the transposon and the adjacent genomic DNA and also helps the excised transposon integrate at a new site

Question 43

How are direct repeats created?

Answer 43

Transposase enzyme recognises inverted repeats and catalyses removal of the TE from it's original site Transposase brings together the inverted repeats Transposase cleaves target DNA sequence at staggered recognition sites Repeats created in the same direction and repeated at both ends of element therefore direct repeats

Question 44

How does replicative transposition work?

Answer 44

Transposase catalyses movement of DNA strand carrying the TE to a new recipient site Gap repair synthesis at the previous and new sites produces two double stranded elements with circular molecules known as cointergrant Resolvase separates cointergrant into two separate structures each containing a TE

Question 45

What is the selfish DNA hypothesis?

Answer 45

TE's exist because they contain the characteristics that allow them to multiply within the host cell DNA therefore resembling parasites because they offer no selective advantage to the host

Question 46

What are some possible advantages to transposition?

Answer 46

``` Promote exon shuffling Promote gene duplication New gene combinations New innovative gene functions Success of evolutionary lineage ```

Question 47

How is methylation releated to transposition?

Answer 47

When DNA is methylated, transposition is inhibited meaning just after fertilisation is when most transposition occurs giving way to 'spontaneous' mutations

Question 48

Describe LINEs

Answer 48

1-5 kb in length 20 - 30k copies per genome Probably the source of LTR transposons and retroviruses Reverse transcriptase encoding region on the transcript is translated into an enzyme that preferentially associates with and uses the transcript it came from as a template to produce LINE cDNA RT often stops before it has made a full length DNA copy of the RNA transcript. cDNA is incomplete and forms a second strand which is integrated back into the genome but is dormant

Question 49

Describe SINEs

Answer 49

``` Less than 500 bp length Nonautonomous Can disperse throughtout genome Evolved from small cellular RNA species Form hairpin loops, RT recognises loop as a primer for elongation ```

Question 50

How are human diseases related to transposons?

Answer 50

Retrotransposons can induce mutations by inserting near or within genes. A retrotransposon induced mutation is relatively stable because the sequence at the site of insertion is retained As multiple copies of the elements build up the potential for unequal crossing over increases leading to disease