James polke institute of neurology london 9 th november 2009
This presentation is the property of its rightful owner.
Sponsored Links
1 / 28

James Polke, Institute of Neurology, London 9 th November 2009 PowerPoint PPT Presentation


  • 47 Views
  • Uploaded on
  • Presentation posted in: General

Describe the methodology behind two next generation sequencing platforms; include strategies for enriching target. What will their application be in the diagnostic laboratory and how will they affect service. James Polke, Institute of Neurology, London 9 th November 2009.

Download Presentation

James Polke, Institute of Neurology, London 9 th November 2009

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


James polke institute of neurology london 9 th november 2009

Describe the methodology behind two next generation sequencing platforms; include strategies for enriching target. What will their application be in the diagnostic laboratory and how will they affect service.

James Polke,

Institute of Neurology, London

9th November 2009


Next generation sequencers

Next Generation Sequencers

  • Available platforms:

    • Roche 454 GS FLX

    • Illumina Genome Analyser II

    • ABI SOLiD 2.0

    • Helicos HeliScope

  • Focus on Roche and Illumina

    • Most popular


Roche 454 sequencing

DNA is fragmented, linkers attached and bound to beads in emulsion: One fragment per bead.

PCR occurs in emulsion droplets, unique sequences attached to each bead are amplified with common primers.

Roche 454 Sequencing

  • Emulsion PCR followed by pyrosequencing

    • Emulsion PCR:


Roche 454 sequencing1

Roche 454 Sequencing

Emulsion is broken. Individual beads are deposited into wells on a fibre-optic slide and pyrosequencing commences.

  • Pyrosequencing:

dNTPs are sequentially added to the slide, light generated by extension is detected: Extension generates PPi. PPi+APS = ATP (ATP-sulfurylase). ATP + luciferse = light

(APS = Adenosine 5' phosphosulphate)


Roche 454 sequencing2

Roche 454 Sequencing

  • Long reads lengths

  • Short run time (10 hrs)

  • Faster but less capacity – less data per £

  • Problems with homopolymers

    • Long stretches of the same base are incorporated in the same step, this results in increased fluorescence, but fluorescence for stretches of >3-4 bases aren't necessarily proportional to length

  • Plate can be divided into 2,4,8 or 16 distinct regions, but this reduces capacity.


Illumina genome analyser

Illumina Genome Analyser

  • 'Bridge PCR' followed by reversible dye-terminator sequencing

  • DNA is fragmented and adapters are added to each end.


James polke institute of neurology london 9 th november 2009

Each cluster has ~1000 copies of the sequence


Illumina genome analyser1

Illumina Genome Analyser

  • Short read lengths – not good for repeat-rich sequences. ~75bp now being quoted.

  • 2.5 days to run

  • Flow cell is divided into 8 channels


Techniques to enrich targets

Techniques to Enrich Targets

  • Illumina (and ABI) are compatible with Agilent SureSelect system:

    • SureSelect DNA Capture Array – Chip based method for smaller studies.

    • SureSelect Target enrichment. Tube-based: Biotinylated RNA 'bait' molecules attach to target sequences, pulled out by attaching to streptavadin-coated magnetic beads. DNA/RNA washed off beads and RNA is digested.


James polke institute of neurology london 9 th november 2009

Techniques to Enrich Targets

  • Data from SureSelect Target Enrichment system

  • Enriching biases towards wt sequences. Sequencing an individual know to be heterozygous for 20000+ SNPs following enrichment:


Techniques to enrich targets1

Techniques to Enrich Targets

  • Roche: Nimblegen sequence capture chips.

    • Up to 5Mb of sequence can be targeted

  • Targeting needs to be 100% for diagnostics

  • 96/97% detection rate not good enough for diagnostics


Techniques to enrich targets2

Techniques to Enrich Targets

  • Long PCR – Used by Graham Taylor to provide templates for breast cancer sequencing on an Illumina GAII. Barcoding allowed 10 patients to be sequenced in a single channel at once (one out of 8, channels – 80 patients at a time?)


Techniques to enrich targets3

Techniques to Enrich Targets

  • RainDance Technologies

  • Microfluidics platform using emulsion droplets in water. A primer library emulsion is made with each droplet containing a unique primer pair. A separate emulsion is made that contains DNA and PCR reagents in droplets. Primer and DNA/PCR droplets are merged 1:1, all droplets can then by multiplexed in the same tube.

  • Promoted to avoid bias seen in chip based methods. Though presumably sensitive to primer binding SNPs


James polke institute of neurology london 9 th november 2009

Some methods are better than others

  • Circularisation/PCR based methods:

None better than 90% capture

ten Bosch and Grody (2008) Journal of Molecular Diagnostics10 484-492


Effect on service

Effect on Service

  • Potentially massively increased throughput but more interpretation and reporting challenges.

  • Higher total cost but lower cost per base call

  • Faster service: Multiple genes can be analysed for each patient at once

  • Avoids mutation detection

  • Easier to look at more – promoter/intronic sequence

  • Paired-end sequencing for CNV/rearrangements

  • More quantitative than Sanger sequencing – detection of low level somatic mutations in cancers/mitochondrial heteroplasmy

  • Applicable to bisulphite sequencing – for imprinting/promoter analysis

  • Better than array CGH – can detect balanced and unbalanced structural variants

  • Private WGS may become a reality, who will interpret the data?


Drawbacks of next gen platforms

Drawbacks of Next Gen Platforms

  • Lower accuracy than Sanger sequencing, but countered by sequencing to great depth

    • Proof-reading polymerases?

  • Lots of pre-sequencing lab work required to fragment DNA and attach adapters. Also steps for barcoding and enrichment.

  • Set up and running costs ($400k-$550k per machine, $3k-$4k per run)

  • Cost of data storage:

    • Running an Illumina 2-3 per week will generate enough data to require $100,000 of hardware per year to store, access and back up the data. Cheaper delete data, to store DNA and repeat analysis if necessary….*

  • Software/bioinformatics to analyse sequence.

  • Too much information… Soon it may be more cost effective to sequence the whole genome than to sequence selected targets.

    • Ethical and interpretive problems

  • Surely there's a market for lower throughput devices?

*ten Bosch and Grody (2008) Journal of Molecular Diagnostics10 484-492


References

References

Roche, Illumina and ABI websites

Nimblegen

Agilent

RainDance Technologies

Reviews:

Tucker et al Am J Hum Genet 2009 85(2): 142-54

Voelkerding et al Clin Chem 2009 55(4): 641-58

Ansorge N Biotechnol 2009 25(4): 195-203

ten Bosch and Grody J Mol Diag 2008 10(6) 484-492


  • Login