Lesson 2 Flashcards
What are the 3 steps of the basic sequencing workflow?
Sample preparation, detection, and analysis
What are the 4 steps of library template preparation?
DNA fragmentation, end repair, da tailing and adapter ligation
What are the 2 reasons why DNA fragmentation is absolutely necessary?
It is impossible to efficiently amplify molecules longer than 1000 bp and NGS sequencers are not able to sequence long reads
What is the most used technique for DNA fragmentation?
Sonication
What is characterized by the addition of a phosphate at the 5’ end of the molecule or filling by DNA polymerase?
End repair
What is added in da tailing?
A nontemplate A at the 3’ end of each strand
What is a sequence which allows for the hybridization of DNA fragments to the flow cell of the sequencer?
Adapter
What is attached to the flowcell chip?
DNA oligomers complimentary to the acceptor sequence
Describe a single index adapter
DNA is inserted in the center surrounded by 2 adapter sequences called p5 and p7 and a 6 letter “barcode” called the I7 index
Describe a dual index
The same as a single index but with 2 indexs added, one at the 5’ end and the other at the 3’ end when the fragment needs to be sequenced from both sides
Describe a dual index UMI
Same as a duel index but with a UMI (a particular barcode not detected in the sample identifying with a molecule or a cell
What type of pcr does ion torrent sequencing use?
Emulsion pcr
What type of pcr does illumina sequencing use?
Polony pcr
When performing emulsion pcr what do the oil droplets created contain?
The fragment, primers necessary to perform the pcr, enzymes + cofactors, and a bead coated by oligos complimentary to the the adapter sequence
What is sequencing depth?
The number of reads obtained
What is the Max read length for ion torrent sequencing?
200 bp
What is the throughput in ion torrent sequencing?
The number of bases you are going to acquire
What is ion torrent sequencing based on the addition of?
A new nucleotide
When is the new nucleotide added in ion torrent sequencing?
During the synthesis of the complimentary DNA strand
What specifically are the wells of an ion torrent chip detecting?
Defects a change in pH due to the release of H+ ions occurring after the incorporation of the nucleotide inside the DNA
What does polomy pcr stand for?
Polymerase colony
What type of sequencing is colony pcr?
Sequencing by synthesis
Describe sequencing by synthesis (polony pcr)
The DNA polymerase amplifies the molecule and if nucleotides are coupled with fluorphores, it is possible to read the sequence
What are two scenarios where colony pcr is useful?
When there are places in the genome with long dimer repeats and to determine now many base pairs are between the 1st and 2nd reads
What is the main difference between 2nd and 3rd generation sequencing?
3rd generation sequencing is able to achieve a much higher read length compared to NGS
What are the two main 3rd gen sequence companies?
Pacific biosciences and Oxford nano por technology
What is unique about pacific biosciences?
Augmentation is still required but no amplification is needed
List the 4 steps of oxford nanopore sequencing:
1) one protein unzips the DNA helix into 2 strands
2) a second protein creates a pore in the membrane and holds an “adapter” molecule
3) a flow of ions through the pore creates a current → each base blocks the flow to a different degree, altering the current
4) the adaptor molecule keeps bases in place long enough for them to be identified electronically
What are the main advantages of nanopore sequencing?
There is no need to amplify the molecules and since no PCR required, it could lead to the achievement of a huge read length
What is the main issue in nanopore sequencing
During BAs coding there is a high % error (10%) due to the use of ai
