3 Building Sequencing Libraries Flashcards
(21 cards)
What is the general workflow of preparing seqeuncing libraries
- Nucleic acids purification (DNA or RNA): also is a way of fragmentation
- Fragmentation
- End repair: use enzymes to repair ends of fragmented DNA
- Adapter addition: adding an adapter to the end of the fragmented DNA that can bind to flow cells , also can be used to add genetic barcodes that lets you multiplex libraries to save sequencing power
- Amplification (optional): Turing a little bit of Nucleic acid to a lot, to have enough material for seqeuncing
- Library clean up and quantification
- Normalization:
What the most difficult step in making a seqeuncing library
Normalization: have to accurately quantify the amount of DNA present is the sample and how much is being loaded
How are the DNA purification steps done
Lyse cells
Chaotropic salt to cause protien denaturation
Silica based column, wash with alcohol buffer
Elute
What are the steps in making illumina seqeunce lobaries “trueseq”
- DNA isolation
- DNA fragmentation: using enzymes that randomly fragment, sonication, chemical, or purification also fragments it
- Size selection: using gel electrophoresis get size you want
- End repair and phosphorylation
- A tailing
- Adapter ligation: adding assymetric Y adapters that are probes with complimentary seqeuncing to the probes in the sequencer chip
- PCR amplification: to get enough DNA for seqeuncing
- Library quantification and screening
How is end repair done
Via T4 DNA Polymerase
It blunts (either but chewing back or filling in the fragments)
5-3 fill in activity
Or 3-5 exonuclease activity to chew back
How is end phosphylation done
T4 polynucleotide kinase
The 5’ end has an oh, need to be phos
T4 polynucleotide kinase uses ATP to add the gamma phos to the 5’ end
Whag is the Klenow and ex Klenow fragment
Use in TA cloning
The exo-Klenow fragment is derived from e.coli dna pol I
DNA pol I has 5-3 polymerase and exonuclease activity and 3-5 exonuclease activity
The Klenow frangment is a big fragment of DNA pol I that’s made when you treat DNA pol I with subtilisin: loses it 5-3 exonuclease activity
Exo-Klenow fragment: Klenow fragment has a mutation the stops it’s 3-5 exonuclease activity
So now eco Klenow only has5-3 polymerase activity
Whag is A tailing and how is it done
After end repair and phosphorylation
The exo Klenow fragment adds a non template A and the 3’ end of the DNA
The addition of the A allows y adapter ligation through DNA ligase
How is y adapter addition done
After a tailing, e.coli DNA ligase ligate the assymetric Y adapters to the ends of the DNA
The you use assymetric PCR to amplify the dna with the adapter
Assymetric PCR because the primers are added in different ratio to amplify one strand more than the other
What is transposition in vitro
Have donor dna with a dna seqeunce you want in another dna strand
The transposomes called the Tn5 transposomes cut that piece of donor DNA and add it to the other strand
What are transposomes used for
To do tagmetation: quickly way to add adapter
The transposomes can insert a stand of dna into another strand that you have in vitro
So they can randomly fragment the dna by inserting adapters : making it so fragemntage and taggin happens at the same time
If mix the transposomes with the dna and the adaptors it can do the entire first few library preps steps all in one cause now you have fragmented dna with adaptors attached
Also quicker
How does PCR limits the ability to finish a bacterial genome
Need to know GC content , if don’t know GC content you can’t optimize the primers
PCR bias: even if tagemtation everywhere, the PCR will amplify certain dna fragments over others based on melting temp (thermodynamic bias)
Can’t know if the primers you made also bind somewhere else in The seqeunce, normally see many diff bands in gel, but PCR bias makes discrete bands
How are the drawbacks to PCR solved
By PCR free library prep
What is good about PCR free library prep
Don’t have to use PCR at all of amplify the sequence
Eliminated bias inherent in PCR and give as much coverage as possible
What the drawback to using PCR free approach over PCR to amplify
might not have enough DNA to do PCR free approach, if don’t have enough starting material you can’t do it
What is SPRI
How does it work
Solid phase reverse immobilization
It’s a clean up step after the library is made , inexpensive
Have prepared DNA have rna and impurites (enzyme, protien), want just DNA
Works :
- Add pherromagentic beads to the tube
- bead binding: DNA binds to the beads, impurites left in solution
- magnetic separation: Magnet pulls the bead with the DNA attached and leave everything else (clear solution, brown dna + beads at bottom of tube
- washing: add buffer to wash away impurites, remove impurities solution
- elution: remove dna from magnetic beads by putting them in a water buffer, dna gets resolublized and released into that buffer, and DNA/RNA only left in solution
What is automated electrophoresis
How does it work
What’s the output
Uses bioanalyzer or Tapestation
Provides DNA size distribution and concentration :
- Do Nucleic acids quantification, and characterization of the size fragments in the mixture in a dna seqeunceing library
- Asses dna and rna quality
Works:
- a matrix material is mixed up and goes inside of wells in the device
- inside each of the wells the matrix is solidified
- pipette your sample to analyze into the well
Output:
- want to see not just how much dna is present but also the size distribution of the fragments you made
- you use the size distribution to calculate the number free ends of the dna that you made (counting molecules) which is important for how you load things onto the illumina flow cell
- ## this then gives you an idea of the average insert size
What size of dna would typically be made in a library’
300bp on average
Bc seqeunce kit have 150-300 cycle which lets you sequence 75-150 bp from either side (total of 300 bp seqeunced)
What does the plot from automated electrophoresis look like
Have standards that are built into the flow cell : one small and one large
Way to determine the concentration and size distribution of the DNA in your library sample
Why would you not want to use the 260/280 method Ive Tthe automated electrophoresis
Doesn’t give Size distribution
Also 260/280 is quick and not precise , okay for cloning but not for quantification of libraries
Fluorimetery ways are also better to quantify Nucleic acids
