Genomes and Sequencing Flashcards
What do you do before you can start to sequence?
Extract DNA and fragment it
What is shotgun sequencing?
Splitting up DNA into fragments randomly to be sequenced
How is shotgun sequencing achieved?
Sonifaication
What is the best sequencing method to use in reality?
Illumina
What type of PCR goes along with Illumina?
Bridge PCR
Why is Illumina the best to use?
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
Why shouldn’t you discuss all of them?
Because that would make it too fragmented and it wouldn’t work in real life
What is an amplicon?
A PCR product
What should your target coverage be and why?
30x because above that you probably can’t improve the error rate anymore
What should your length be?
At least 15 - 20 base pairs
How does Illumina work?
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.
Outline your methodology for sequencing a genome
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
Describe the assembly process
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
What is meant by coverage?
The number of times a section of the genome is represented in the sequenced fragment
What is meant by identity?
The percentage of the reads that agree with the same nucleotide
What are identity and coverage used for?
To discount errors and differentiate between errors and heterozygosity. 50% or close indicates heterozygosity
What is bin size?
When boundaries are set to distinguish between statistical variation and error
What is genomics?
The study of all the genes of a cell or tissue at the DNA, mRNA or protein level
What is comparitive genomics?
The study of the relationship of genome structure and function across different biological species or strains
What is functional genomics?
The study of gene and protein functions and interactions utilising the data produced by genome projects
What are the 2 methods of gene duplication?
Nonhomologous recombination and retrotransposition
What is unequal crossing over?
Two non homologous DNA double helices align and undergo recombination which is greatly facilitated if the two strands already contain repeat units
What are the 4 levels of duplication?
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
What is a multigene family?
A set of genes descended by duplication and diversification from one ancestral gene
Can be tandem or dispersed
What are pseudogenes?
The decomposing remnants of either failed duplication events, transposition genes or disabled genes
Unnecessary copies of genes
What are tandem arrays?
The same sequence repeated over and over
What is the crossover fixation model?
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
What are Hox genes?
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
How did Hox genes arise?
Gene duplication
What is the immunoglobulin superfamily?
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
What is site-specific recombination?
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
What are transposable elements?
DNA segments that transpose themselves
Describe what happens when transposition occurs to an already replicated recipient site
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
Describe what happens when transposition occurs to an unreplicated recipient site
Transposition occurs to parental strand before replication fork
Completion of replication
Net increase in number of Ac elements
Name the 4 classes of transposons
LINEs
SINEs
Long Terminal Repeat retrotransposons
DNA transposons
What is simple transposition?
Cut and paste
Bacteria and eukarya
What is replicative transposition?
Copy and paste
Uncommon
Bacteria
What is retrotransposition?
Common in eukarya
Where are direct repeats?
Found within the host DNA
Where are inverted repeats?
At the ends of most transposable elements
Describe the structure of a transposon
Inverted repeats at the ends
Encode transposase to recognise inverted repeats
How does transposase work?
Cuts at the borders between the transposon and the adjacent genomic DNA and also helps the excised transposon integrate at a new site
How are direct repeats created?
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
How does replicative transposition work?
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
What is the selfish DNA hypothesis?
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
What are some possible advantages to transposition?
Promote exon shuffling Promote gene duplication New gene combinations New innovative gene functions Success of evolutionary lineage
How is methylation releated to transposition?
When DNA is methylated, transposition is inhibited meaning just after fertilisation is when most transposition occurs giving way to ‘spontaneous’ mutations
Describe LINEs
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
Describe SINEs
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
How are human diseases related to transposons?
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
