in Silico Primer Design and Simulation for Targeted High Throughput Sequencing

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• in Silico Primer Design and Simulation for
  Targeted High Throughput Sequencing

I519 FALL 2010 Adam Thomas, Kanishka
Jain, Tulip Nandu
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BACKGROUND

• Major Milestone
• Molecular structure of DNA
• Human Genome Project
• High-Throughput Sequencing (HTS)
• HTS transformed common experiments on single
  genes to entire genomes
• Low cost
• Multiple samples in every run (Eg. 454 Sequencer
  can sequence 400-600Mb)

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BACKGROUND

• Primers are a short stand of nucleotides that
  serve as the starting point of DNA synthesis.
• Approximately 20-25 nucleotides.
• Used to determine the DNA strand that needs
  amplification.
• Complement of DNA strand.

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PCR

• Polymerase Chain Reaction
• Technique to amplify a small region of DNA
• 3 step process
• Denaturation,
• Annealing and
• Extension.
• Process repeated for approximately 30 to 40
  cycles.

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PCR

• Denaturation

Heat (approx 90C) separates double strand into
two single strands
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PCR

• Annealing

Primer binding to individual strands (occurs at
45 to 60C)
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PCR

• Extension

Temperature raised to 72C and the Tag DNA
polymerase enzyme is used to replicate DNA strands
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PCR

• End of First Cycle

Process repeated for approximately 30 to 40
cycles.
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CURRENT PROCESS
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CURRENT PROCESS

• Primer3 used to create primers using PCR.
• The primers then need to be validated. Validation
  is performed by simulation, alignment and
  re-assembly.
• MetaSim is used to simulate PCR to create
  expected amplicons.
• CAP3 is used for re-assembly of simulated
  sequences.
• BLASTing the simulated sequences against the
  original sequence give a fairly accurate measure
  of how well the primers will perform.

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ISSUES FACED WITH CURRENT PROCESS

• Each tool uses different file inputs and outputs.
• Users have to manually convert file formats to
  use in each tool.
• None of the tools up till now can integrate all
  of the functions and give high throughput
  analysis.

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GOAL

• Integrate the whole process involved in the High
  throughput sequencing experiment and keep track
  of the parameters that are enter or changed.

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OBJECTIVES

• A way to visualize the primers and amplicons in
  relation to the genome and be able to edit the
  primers manually and see how that affects the
  simulation.
• Optimization of the high-throughput process by
  minimizing the number of reads needed by the 454
  process and still be able to assemble the
  sequence.
• Validation of the simulated amplicon reads to see
  whether the predicted simulation is in order and
  rectify the problem.

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PROPOSED SOLUTION
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PROPOSED SOLUTION

• Solution can be broken into two major components
• Creation of overlapping amplicons when amplified
  by PCR
• Validation of primers effectiveness in silico.
• Automation of the above mentioned solution.

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VISUALIZATION TOOL

• GBrowse
• Popular and open source.
• Well defined plugin architecture.
• Plugin to design primers using Primer3 already
  available.

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PRIMER DESIGN

• PrimerDesign.pm plugin already exists for
  GBrowse. Design primers using Primer 3
• Designed to only amplify one specific region of
  DNA with as few primers and no overlapping
  amplicons.
• Tweaked to take two additional input parameters
  Amplicon Overlap and Max Amplicon Length.
• Once primers are created using GBrowse, the
  primers are output into a Featured File Format
  (FFF)

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PRIMER VALIDATION - SIMULATION

• Simulation performed using MetaSim.
• MetaSim
• Generates sets of synthetic reads or mate-pairs
  based on adaptable sequencing error models (e.g.
  for Sanger chemistry, Roche's 454 and Illumina
  (former Solexa).
• Can be controlled via graphical user interface or
  in command line mode.

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SIMULATION

• Function written in Perl to invoke MetaSim using
  command line option.
• Algorithm
• Read FFF file. Extract primer coordinates.
• Extract sequence from the original sequence.
• Run MetaSim simulation using command line
  options.
• Each sequence generates its own FASTA sequence
  file with multiple sequences.

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ASSEMBLY

• Perl function written to invoke CAP3 using its
  command line interface.
• Each file generated from the MetaSim simulation
  is input into CAP3 which then assembles the
  contigs.

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ASSEMBLY

• CAP3.
• Input simulated sequences as FASTA file.
• CAP3 is a sequence assembly program that allows
  users to assemble a set of short contigs.
• Takes an input a file of sequence reads in FASTA
  format.
• If header contains a dot (.), CAP3 requires
  that the names of reads sequenced from the same
  subclone contain the same substring up to the
  first dot.
• Can be invoked using a command line interface.

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BLAST

• Assembled contigs are then BLASTed against the
  original sequence to validate.
• GBrowse accepts the assembled sequence and BLASTs
  against the original sequence.
• This plugin requires 4 steps
• Exporting assembled contigs and original sequence
  from Gbrowse.
• Creating a BLAST database.
• BLASTing the contigs against the sequence.
• Importing result back into GBrowse.

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DEMO
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QUESTIONS