Sequencing

Question 1

Key NGS steps?

Answer 1

• library prep, to fragment samples and mod w/ custom adapter sequence
• capture step (optional), to pull out region of interest for sequencing
• amp, to prod DNA clusters each originating from a single DNA fragment
• sequencing, each seq is a read of 50-300bp
• alignment, to ref seq
• variant calling, identities diffs
• variant annotations, ie. likely effect

Question 2

How is automated Sanger seq carried out in NHS?

Answer 2

• PCR w/ fluorescent chain-terminating ddNTPs, plus dNTPs
• incorp ddNTP terminates seq, get group products stopping at primer +1, 2 etc.
• ran in single capillary gel electrophoresis w/in sequencing machine, sep by size
• computer reads each band, using fluorescence to identify ddNTP, by laser exciting tag
• get trace differential (chromatogram), takes 1st trace away from 2nd, so see what diff is in peak height and area
• -> no diff = straight line, diff = bubble
• -> heterozygote has 2 peaks in same location at 2 height (as 2 seqs)
• -> sudden change after certain point likely fs (ie. all much lower and some changed)

Question 3

How does NGS differ from Sanger seq?

Answer 3

• DNA seq libraries clonally amplified in vitro by PCR, rather than template prep by PCR
• DNA seq by synthesis w/ add of nts to complimentary strand, rather than ddNTPs
• products spatially segregated, rather than physically w/ gel tech
• much more data prod and much quicker
• can look at larger gene panels and whole genomes etc.
• could replace microarrays, chrom analysis, dosage, meth analysis, southern blotting

Question 4

How does Illumina work?

Answer 4

• cut gDNA into 200-600bp fragments
• add adapters
• DNA fragments which bind adapters are made ss
• adapters bind oligos on flow cell surface
• unlabelled nt bases and DNA pol added to lengthen and join DNA seqs
• adapter seq at other end binds another type of oligo on surface and creates ‘bridges’ of ds DNA on flowcell surface (by seqs folding over and hybridising to oligos)
• in situ PCR = bridge amplification –> amplify original DNA to form small clusters of DNA w/ same seq
  dsDNA bridges broken down to ssDNA w/ heat
• primers and fluorescently labelled bases added to flowcell
• primer binds DNA being seq and allows DNA pol to bind
• DNA pol adds bases to DNA
• lasers used to activate fluorescent label and camera detects this fluorescence
• each base gives off diff colour

Question 5

What is the main problem w/ Illumina?

Answer 5

• reads are too short, due to phasing

- this means quality of reads reduces w/ length as chance of random base not being incorp and cause this to lag behind

Question 6

What is a gene panel?

Answer 6

• collection of genes to be sequenced together, which are usually linked by common biological pathways, or known disease associations