Sequencing platforms

Question 1

Which sequencing platform:

has 10,000-15,000 bp reads
Produces ~50,000 reads per flow cell
uses phospholinked nucleotides (fluoresce on term. PO4)
uses zero-mode-waveguide cylindrical chambers
can multiplex?

Answer 1

PacBio (Single-moleculre real-time sequencing)

Question 2

Which sequencing platform has the longest read length?

Answer 2

PacBio (Single-moleculre real-time sequencing)

10-15k reads

Question 3

Which sequencing platform:

has reads up to 400 bp long
is based on Sanger seq.
detects the release of H+ ions during the sequencing reaction (changes in pH) as wells are flooded with a single nt?

Answer 3

Ion torrent

Question 4

How long are the reads produced by 454 sequencing?

Answer 4

700 bp

Question 5

Which sequencing platform:

Produces 700 bp reads
uses DNA amplified on beads in water droplets in an oil emulsion PCR
uses luciferase to generate light for the det. of nts during the amplification

Answer 5

454 pyrosequencing

Question 6

What is another name for Illumina sequencing?

Answer 6

Sequencing by synthesis

Question 7

How many bp can be produced in an Illumina run?

Answer 7

50-300 bp (depending on platform)

Question 8

How are samples prepped for Illumina sequencing?

Answer 8

Fragment DNA (300-1500 bp)
attach sequencing primers and adapters

Question 9

How are clusters generated for Illumina sequencing? (name and 6 steps)

Answer 9

Clonal amplification through bridge amplification

1. library ssDNA fragments to a glass slide w/ lanes (flowcell) via attached oligos
2. ssDNA strand flods over to a second oligo (form bridge)
3. polymerases generate dsDNA (bridge amplification)
4. bridge is denatured, get two attached ssDNA clones for each bridge amplification
5. repeated over and over for all fragments (parallel amplification)
6. R strands are cleaved off, leaving only the forward strand for sequencing (this process will be reversed for F strands to result in paired-end sequencing)

Question 10

Why are Illumina reads so short?

Answer 10

Phase errors during massively parallel sequencing (signals can get out of sync causing errors, esp. at ends)

Question 11

How does Illumina sequencing of clusters work? (2 steps)

Answer 11

1. Competitive binding of fluorescently-labelled nts to the clusters
2. Detection of cluster signal using a light source

Question 12

Which sequencing platform uses:

50 bp read length
clonal amplification in PCR emulsion
Beads attached to a glass slide
Fluorescent labelled by read in 5 bp for 5 rounds

Answer 12

SOLiD sequencing

Question 13

What is another term for Sanger sequencing?

Answer 13

Chain termination method

Question 14

What are the two main shearing methods?

Answer 14

Sonification (high sound energy)

Nebulization (forcing through a small hole in a nebulizer using pressurized gas)

Question 15

What sequencing accuracy does a Phred score of 20 represent? 30? 40?

Answer 15

20: 99%
30: 99.9%
40: 99.99%

Question 16

What is the cost per million bp for Illumina sequencing?

Answer 16

$0.13