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in Silico Primer Design and Simulation for Targeted High
Throughput Sequencing
in Silico Primer Design and Simulation for Targeted High
Throughput Sequencing
I519 – FALL 2010Adam Thomas,Kanishka Jain,
Tulip Nandu
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BACKGROUNDBACKGROUNDn Major Milestone
n Molecular structure of DNA
n Human Genome Project
n High-Throughput Sequencing (HTS)
n HTS transformed common experiments on single genes to entire genomes
n Low cost
n Multiple samples in every run (Eg. 454 Sequencer can sequence 400-600Mb)
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BACKGROUNDBACKGROUNDn Primers are a short stand of nucleotides that
serve as the starting point of DNA synthesis.
n Approximately 20-25 nucleotides.
n Used to determine the DNA strand that needs amplification.
n Complement of DNA strand.
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PCRPCRn Polymerase Chain Reaction
n Technique to amplify a small region of DNA
n 3 step process:
n Denaturation,
n Annealing and
n Extension.
n Process repeated for approximately 30 to 40 cycles.
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PCRPCRn Denaturation
Heat (approx 90°C) separates double strand into two single strands
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PCRPCRn Annealing
Primer binding to individual strands (occurs at 45 to 60°C)
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PCRPCRn Extension
Temperature raised to 72°C and the Tag DNA polymerase enzyme is used to replicate DNA strands
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PCRPCRn End of First Cycle
Process repeated for approximately 30 to 40 cycles.
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CURRENT PROCESSCURRENT PROCESS
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CURRENT PROCESSCURRENT PROCESSn Primer3 used to create primers using PCR.
n The primers then need to be validated. Validation is
performed by simulation, alignment and re-assembly.
n MetaSim is used to simulate PCR to create expected
amplicons.
n CAP3 is used for re-assembly of simulated sequences.
n 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 PROCESSISSUES FACED WITH CURRENT PROCESS
n Each tool uses different file inputs and outputs.
n Users have to manually convert file formats to use in each tool.
n None of the tools up till now can integrate all of the functions and give high throughput analysis.
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GOALGOAL
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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OBJECTIVESOBJECTIVES
n 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.
n 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.
n Validation of the simulated amplicon reads to see whether
the predicted simulation is in order and rectify the problem.
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PROPOSED SOLUTIONPROPOSED SOLUTION
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VISUALIZATION TOOLVISUALIZATION TOOLn GBrowse
n Popular and open source.
n Well defined plugin architecture.
n Plugin to design primers using Primer3 already available.
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PRIMER DESIGNPRIMER DESIGNn PrimerDesign.pm plugin already exists for GBrowse. Design
primers using Primer 3
n Designed to only amplify one specific region of DNA with as
few primers and no overlapping amplicons.
n Tweaked to take two additional input parameters: Amplicon
Overlap and Max Amplicon Length.
n Once primers are created using GBrowse, the primers are
output into a Featured File Format (FFF)
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PRIMER VALIDATION - SIMULATION
PRIMER VALIDATION - SIMULATION
n Simulation performed using MetaSim.
n MetaSim:
n 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).
n Can be controlled via graphical user interface or in
command line mode.
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SIMULATIONSIMULATIONn Function written in Perl to invoke MetaSim using
command line option.
n Algorithm:
n Read FFF file. Extract primer coordinates.
n Extract sequence from the original sequence.
n Run MetaSim simulation using command line
options.
n Each sequence generates its own FASTA
sequence file with multiple sequences.
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ASSEMBLYASSEMBLYn Perl function written to invoke CAP3 using its
command line interface.
n Each file generated from the MetaSim
simulation is input into CAP3 which then
assembles the contigs.
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ASSEMBLYASSEMBLYn CAP3.
n Input simulated sequences as FASTA file.
n CAP3 is a sequence assembly program that allows users to assemble a set of short contigs.
n Takes an input a file of sequence reads in FASTA format.
n 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.
n Can be invoked using a command line interface.
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BLASTBLASTn Assembled contigs are then BLASTed against the original
sequence to validate.
n GBrowse accepts the assembled sequence and BLASTs against the original sequence.
n This plugin requires 4 steps:
n Exporting assembled contigs and original sequence from Gbrowse.
n Creating a BLAST database.
n BLASTing the contigs against the sequence.
n Importing result back into GBrowse.
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DEMODEMO
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QUESTIONSQUESTIONS