sequencing

Question 1

What is needed for DNA replication?

Answer 1

• Template (DNA)
• Primers
• Nucleotides
• Enzyme cofactor (MgCl2)
  Polymerase
  DNA duplex unwinding machinery (helicase)

Question 2

What is needed for a PCR reaction?

Answer 2

• Template (DNA
• Primers
• Nucleotides
• Enzyme co-factor
• Polymerase - HEAT STABLE!
• Thermal cycler

Question 3

How should a primer be designed?

Answer 3

• Length: 17-30bp
• Melting temperature (Tm) : 50-55C
• GC-content: ~50%
• 3’ nucleotides: G or C

Question 4

How does Sanger sequencing work?

Answer 4

• DNA is denatured (heat)
• Primers anneal to the 5’ strand
• 4 reactions with different labelled dNTPs are set up
• One modified nucleotide (ddNTP) is added - causes chain termination.
• DNA sizes are separated on gel.
• All strands begin with the same sequence, end on different dNTPs.

–> Piece together a whole sequence!

Question 5

What are the limitations of Sanger sequencing?

Answer 5

• 500-800bp/read

- Secondary structures in the DNA can give sequence errors

Question 6

What is the Illumina technology?

Answer 6

A sequencing method that can be used for either whole genome or regional sequencing, as well as small RNA discovery, methylation profiling, etc.

Based on sequencing by synthesis.

Question 7

What is the basic NGS workflow?

Answer 7

• Library preparation (random fragmentation of DNA or RNA + ligation of adaptors)
• Cluster generation (clonal amplification)
• Sequencing (qequencing by synthesis (SBS))
• Data analysis (match to a reference genome)

Question 8

What is “sequencing by synthesis”?

Answer 8

One flourescently labelled nucleotide at a time attaches complementary to the DNA strand

• -> gives fluorescent signal upon attachment from excitation of light
• -> creates a color pattern depending on the nucleotide attaching to the sequence

Question 9

How is an adaptor constructed?

Answer 9

P5/7 regions = complementary to the oligo lawn.

Primers = starts synthesis of a complementary strand.

Index = sequence used for identification of a library.

Question 10

How does cluster amplification work?

Answer 10

Strand attaches to oligo via P5/7 region

• -> bent over, attaches to P7/5
• -> is primed and extended
• -> denaturation
• -> strands are continuously used as temples for creating new, complementary strands (similar to PCR)

Question 11

What is the meaning of:

• Read
• Paired-end read
• Mate-pair read
• Insert size
• Contig
• Scaffold
• Coverage

Answer 11

• Read = a sequenced piece of DNA
• Paired-end read = sequencing both ends of a fragment
• Mate-pair read = sequencing both ends of a LONG fragment
• Insert size = fragment length
• Contig = overlapping segments representing a consensus region
• Scaffold = contigs separated by a gap of known length
• Coverage = the number of time a region is covered by reads