next generation sequencing Flashcards
why do we sequence the whole genome
-biodiveristy and speciation
-diversity within a species
-biology of organism
how is DNA extended
through 3’0H group of the pentose sugar by 5’ phosphate of free nucleotide. Phosphodiester bond formed and diphosphate released.
what are the 3 main stages of PCR
-denaturation
-anneal primer
-extend new strand by incorporating dNTPs
what are the main steps are Sanger sequencing
dna fragenatation, clone into vectors, transform bacteria, grow and isolate vector DNA. Sequence librbay and assemble contiguous fragments
how many base pers can Sanger do per sequence
up to 700
what is bases are included in Sanger sequencing
nucleotides and dideoyxnucleotides- labelled terminators
How does Sanger extend and work out sequencing
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
what anaylses the results of sanger sequencing
capillary electrophoresis and a laser reader
what is the disadvantage of sanger
low output redepth of 1
how is the addition of a base detected in 454 technology
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
what is the main issue with 454 technology
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%
Which method is optimised for small genomes
illumina
what type of DNA shearing would you use for illumina and why
enzymatic and because you use small genomes
what do transposons do
they cut the DNA fragmenting it and then add adapter to the DNA
how long does illumination take
in up to 2 days can get around 10 billion reads
how can you sample pool
can add an index sequence which is 8-12 bp taht allows you to pool mulitiple fragments together.
what do the p5 and p7 so in illumina
allow the DNA to bind to the flow cell
what are the advantages of sample pooling?
-reduces reagent cost
-quicker time around time per sample
what are the disadvantages of sample pooling
Reduced read number per sample
-introduces normalisation step to minimise variation in read number per sample
what is paired end indexed sequencing needed for and which technology uses it
required for gene variation and is used in illumina
what is the advantages of paired end indexed sequencing
-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.
how does paired end indexed sequencing work
one ion the olgios on the floor cell is complementary to the
How does 454 detect the light
Plate is coupled with a fibre optic chip. A CCD camera record the light
What is the size of reads from Pacbio
Much bigger at 10kb to 20kb
What are advantages of pacbio
Bigger read
Can span repeat regions to determine sequences and close up genomics
What type of dna shearing does pac bio
Mechanical: sonofication or g tubules
What is enzymatic shearing used for
Small dna sequences- if too big would cut too small
How does sonificstion work
Fire ultra focused sound waves at different wavelengths at your dna which breaks it to the size you want
What advantage does g tubule have over sonification and disadvantage
Cheaper but has lower throughput
How do g tubules work
They fire DNA through a fine mesh in a centrifuge which breaks down DNA. How fast u spin changed the size of your DNA
What is needed for sample prep after dna shearing in Pacbio
Smart bell formation
-fragment
- repair ends
-ligate adapters
- purify dna
- sequencing
What are the two different sequencing modes in Pacbio and which is better
LS- long sequence reads
CCS- Hugh quality sequencing reads
CCS looks at shorter fragments but does multiple copies so can fix if error
How does Pacbios way of adding fluoroflores differ to illumina
Uses triphospahte linked fluorophores to reduce steric hinderance which allows DNA pol to move quicker
How does pacbio detect fluorescent signals
Zero mode wavelengths hold fluorescent signal and can detect base incorporated despite back ground of other nucleotides
How does pacbio detect flourcence
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
What are the advantages of pac bio
Short waiting time
Long reads
Direct observations
No amplification required
What is the advantages of oxo nanowire
Can sequence extra long reads
No amplification needed
Can use pcr but don’t have to
Can look at DNA and RNA
What is the disadvantage of nanopore
It’s accuracy is in question
90-99%
How does nanopore work
A current passes through the nanopore
When the bases come through they disrupt the ion flow and each base disrupts differently giving differnt reads
What accuracy and error is a factor phred score of 20
99% and 1/100
What accuracy and error rate is a phred score of 30
1/1000 and 99.9%
What could cause a poor distribution per base quality
General degradation of quality over duration of long runs
What do the different colours mean in the phred scores
Colder mean higher quality
Warmer mean Lower quality
What causes low quality scores and what do u do
Imaging problems or technical problems
Need to remove it from down stream so isn’t patty of analysis
How to tel if GC content is bad
There will be a sharp peak where there should be a smooth curve which suggests there could be confinement
What might over representation indicate
Over resperenatation of a sequence indicates taht the libabry might be contaminated
Why are there gaps in de novo assembly
Because of repetive sequences
How is reference mapping done
Against a reference genome
What does reference mapping do
Allows you to identify single nucleotide polymorphisms, insertions and deletions
What programs do reference mapping
Bwa
Bowtie
What program allows manual inspection of sequences and mapped reads
Artemis
What is the difference between synomous snp and non synonyms snp
Synmous - no change to aa
Non synmous- change to aa
What if the difference between missense and non sense snps
Missense- differnt as coded
Nonsense- results in premature stop codon
What is trams
It’s a rapid annotation toon for annoattaion of microbial snp
Searches for multiple nucleotide polymorphisms
