Module 1: Lesson 2 Flashcards
What are “Top Down” strategy characteristics?
microbiological “libraries”, minimal “tiling path” of cloned segments, and minimizing sequencing
What are the types of sequencing technologies?
Maxam-Gilbert sequencing and Sanger sequencing
What is Maxam-Gilbert sequencing?
Chemical sequencing, resulting in chemical cleavage isolated DNA
What is Sanger sequencing?
Chain termination - nucleotide addition stopped by ddNTPs
What is “Bottom Up” sequencing?
Sequencing “assembled” by connecting overlapping information obtained from random sequence “reads”
What are the prerequisites of NGS?
Large-scale Shotgun sequencing “mastered”, independence from cloning, new platforms, new computer algorithms, and advances in computer technology
What is Shotgun sequencing?
A major advance with the use of a “paired-end” approach sequencing both ends of fragments helps orient sequence and validate mapping
Can a whole genome be sequenced using bottom-up methods?
No
Who is Celera?
The commercial competitor to the publicly funded Human Genome Project consortium (pioneering bottom-up sequencing)
How do you sequence by synthesis?
Fluoresce, luciferase, and ion semiconductor (hydrogen detection)
How do you sequence by ligation?
Fluoresce and rolling circle replication (DNA nanoballs)
How do you conduct bridge PCR?
DNA fragments are flanked with adaptors. A flat surface is coated with two primers corresponding to the adaptors. Amplification proceeds in cycles, with one end of each bridge tethered to the surface. Used by Illumina/Solexa.
How is emulsion PCR conducted and who uses it?
Fragments, with adaptors, are PCR amplified within a water drop in oil. One primer is attached to the surface of a bead. 454, Polonator, and SOLiD use it.
What is a pyrosequencing reaction?
Nucleotide incorporation generates light seen as a peak in the pyrogram.
What are NGS sources of error?
Hybrid clusters, inadequate reading depth, and extremes in GC content.
How does nanopore sequencing work?
When sequencing DNA or RNA with nanopores, changes in current caused by the strand as it passes through the pore are recorded.
What is a GEM?
GEM, or a gel bead in an emulsion droplet, is a technology that contains a 16-base barcode unique to the bead and amplification reagents.
How does 10x genomics work with GEM-based technologies?
A microfluidic system partitions and delivers high-molecular-weight DNA to the beads, such that each bead only contains around 10 molecules. The molecular biology of the system is arranged to produce constructs possessing the barcode, which can be sequenced in an Illumina instrument and are ~350 base pairs of gDNA.
What are whole genome approaches?
Whole genome approaches come in two categories: read alignment/mapping and De Novo assembly. Read alignment/mapping pertains to reference sequence and closely related phylogenetic species. De Novo assembly pertains to the single method and combined sequence method.
What are the ways to generate De Novo complete genome sequence?
Hybrid assembly and telomere-to-telomere (T2T) human genome sequence.
