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Sequencing introduction

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by

Sean May

on 22 August 2014

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Transcript of Sequencing introduction

Standard (Sanger) sequencing
Template
Primer
Random ddNTP termination.
Label can be added to the:

Primer
ddNTP –or-
Incorporated dNTPs
“Solexa sequencing”
Series of images taken from www.illumina.com
Solexa sequencing II
“Solexa sequencing”
454 sequencing (images by Roche)
Sample Input and Fragmentation: Genomic DNA or BACs are fractionated into small, 300- to 800-basepair fragments

Library Preparation: Short adaptors (A and B) - specific for both the 3' and 5' ends - are added to each single stranded fragment.

One Fragment = One Bead: Each fragment of the single-stranded DNA library is immobilized individually onto beads in a water-in-oil mixture.

emPCR (Emulsion PCR) Amplification: Each unique fragment is amplified in parallel to several million per bead.

One Bead = One Read: The clonally amplified fragments are loaded onto a PicoTiterPlate device for sequencing. Only one bead per well.

Auto fluidics flows individual nucleotides in a fixed order across the hundreds of thousands of wells containing one bead each. Addition of a nucleotide results in a chemiluminescent signal.
Data overload
Rubber sequencing 2010 estimate
Spotted at Damisha beach Shenzhen (2012)
http://www.sciencemag.org/site/products/posters/SequencingPoster.pdf
Full transcript