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lecture_notes:04-01-2011

Sequencing Technologies, Nader Pourmand

Nader detailed the procedures that were used to generate reads from the banana slug genome, as well as shortcomings that we would need to be aware of while processing the results:

Library Preparation

Size selection

  • On gel, DNA tangling causes size selection to be imprecise.
  • Instruments: SAGE, Caliper

Sequencing Technologies

Sequencing by synthesis (454 and Illumina)

  • Synthesis limits sequencing 5' to 3'

Sequencing by ligation (SOLiD)

Pyrosequencing (454)

Paired tag (Illumina and SOLiD)

In-house

  • Nader's lab uses a method that starts by fragmenting the DNA into long fragments (aiming for ~10k). They attach a unique barcode primer to each fragment and do circular ligation, much like SOLiD. This is followed by circular replication, primer binding, and random cleavage to obtain fragments of different lengths [1]. They sequence both ends of each fragment on the Illumina, obtaining a paired read with its 10k fragment's barcode.
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lecture_notes/04-01-2011.txt · Last modified: 2011/04/03 12:28 by eyliaw