Bioinformatics 14: High throughput sequencing 2

Question 1

What is SMRT sequencing? Why is it novel technology?

Answer 1

SMRT -> Singe Molecule Real-Time

Sequencing by single, natural polymerase molecules
-> incorporation of fluorescently labelled dNTPs captured in real time

Zero Mode Waveguide imaging

Real-time output of 1000s of polymerases captured simultaneously (fast computing)
Revolutionary read length (~4kb)
450Mb in hours (speeeeeeed)

Question 2

How does SMRT sequencing work?

Answer 2

Zero Mode Waveguide (ZMW) contains single polymerase (complexed with template)
- Only bottom of the well is illuminated

nucleotides diffuse in and out of well (microseconds event)
incorporation via polymerase over microseconds (1000x slower)

Real time data collection -> computer detects release of fluorescent label (incorporation event)

Question 3

Pros and cons of SMRT sequencing?

Answer 3

Pros
Single molecule (no amplification) - easy sample prep
Long templates (rolling circle amplification)
Speedy, low cost reagents

Cons
DNA only
Higher error rate
Low no. of features vs 454 + illumina
Late to market

Question 4

Applications of SMRT?

Answer 4

Fast sequencing + long reads useful for quick, complete genome sequencing

Haiti cholera outbreak, Sproutgate

Question 5

What format is NGS data universally stored in? What information does the format give?

Answer 5

fastq

gives sequence AND quality score

quality = ‘Phred’ -> 10 - 1/10 error, 20 = 1/100 error …. so on

Question 6

Other forms of NGS?

Answer 6

Exome sequencing

Nanopore