Module 1: Lesson 2

Question 1

What are “Top Down” strategy characteristics?

Answer 1

microbiological “libraries”, minimal “tiling path” of cloned segments, and minimizing sequencing

Question 2

What are the types of sequencing technologies?

Answer 2

Maxam-Gilbert sequencing and Sanger sequencing

Question 3

What is Maxam-Gilbert sequencing?

Answer 3

Chemical sequencing, resulting in chemical cleavage isolated DNA

Question 4

What is Sanger sequencing?

Answer 4

Chain termination - nucleotide addition stopped by ddNTPs

Question 5

What is “Bottom Up” sequencing?

Answer 5

Sequencing “assembled” by connecting overlapping information obtained from random sequence “reads”

Question 6

What are the prerequisites of NGS?

Answer 6

Large-scale Shotgun sequencing “mastered”, independence from cloning, new platforms, new computer algorithms, and advances in computer technology

Question 7

What is Shotgun sequencing?

Answer 7

A major advance with the use of a “paired-end” approach sequencing both ends of fragments helps orient sequence and validate mapping

Question 8

Can a whole genome be sequenced using bottom-up methods?

Answer 8

No

Question 9

Who is Celera?

Answer 9

The commercial competitor to the publicly funded Human Genome Project consortium (pioneering bottom-up sequencing)

Question 10

How do you sequence by synthesis?

Answer 10

Fluoresce, luciferase, and ion semiconductor (hydrogen detection)

Question 11

How do you sequence by ligation?

Answer 11

Fluoresce and rolling circle replication (DNA nanoballs)

Question 12

How do you conduct bridge PCR?

Answer 12

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.

Question 13

How is emulsion PCR conducted and who uses it?

Answer 13

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.

Question 14

What is a pyrosequencing reaction?

Answer 14

Nucleotide incorporation generates light seen as a peak in the pyrogram.

Question 15

What are NGS sources of error?

Answer 15

Hybrid clusters, inadequate reading depth, and extremes in GC content.

Question 16

How does nanopore sequencing work?

Answer 16

When sequencing DNA or RNA with nanopores, changes in current caused by the strand as it passes through the pore are recorded.

Question 17

What is a GEM?

Answer 17

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.

Question 18

How does 10x genomics work with GEM-based technologies?

Answer 18

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.

Question 19

What are whole genome approaches?

Answer 19

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.

Question 20

What are the ways to generate De Novo complete genome sequence?

Answer 20

Hybrid assembly and telomere-to-telomere (T2T) human genome sequence.