huong le department of molecular & clinical genetics, royal prince alfred hospital click mouse...
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Huong LeDepartment of Molecular & Clinical
Genetics,Royal Prince Alfred Hospital
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Sequence variant detection SOFTWARES
Sequence variant interpretation SOFTWARE
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Seqscape v2.5 Mutation surveyor
◦ AB software◦ Seqscape software using the KB Basecaller
algorithm (v1.2) integrated with the Phred software provide quality value calculation per base.
◦ Seqscape software contains many features such as base calling program
Base calls Measure quality value (QV) Identify pure and mixed bases Trim quality sequence Filter out the poor sequence
sequence assembly, alignment and comparison
Search library for allele match Generate reports
◦ SoftGenetic software is a pioneer in mutation detection from DNA sequence traces
◦ Mutation surveyor software use direct sequence trace comparison method (the anti-correlation method) to locate Heterozygote / Homozygote point and
indels mutations◦ The software remove the effect of base
calling error and reduced the fault positive rate as the rate of human mutation is ~1/1200 or
0.08% ) and basecalling accuracy as Phred score of 18-20 is approximately to 98-99%
◦ Therefore sensitive for detection of rare variants that present as low as 10% of the population using the 2Dir smaller peaks
◦ Robust sequence aligment
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Seqscape and cDNA annotation set up
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Seqscape and cDNA annotation set up
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Seqscape – Genotype Table
c. 681 location
g. 2479065 location
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Trace Comparison The DNA nucleotide peak intensities of traces are normalised and aligned and then the actual traces are compared using the mathematical algorithm called anti-correlation
Reference trace R
Sample trace S
Difference between
the R and S trace green line
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The anti-correlation method◦The differences between reference and
sample traces were calculated and note any found in the mutation electropherogram. At any specific location that reference and sample have the same type of bases and the anti-correlation will be zero otherwise this value will be very high if these bases are different at the same point
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The four numbers on the mutation peak is the score, mutation peak height,
overlapping factor and intensity dropping factor.
mutation peak height (the highest peak in mutation electropherogram)
Overlapping factor indicate the relative shift of the two peaks
(reference/sample) of different colors in the horizontal direction
Intensity dropping factor indicate the vertical peak intensity has dropped relative to the neighboring peaks
Mutation score= -10log(erfc[(s/n)/√2])*dropping
factor*overlapping factorerfc: complementery error
function
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Mutation Surveyor and result output
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Homozygous indel detection and robust alignment based on the three following methods◦ Rough alignment with sequence text: use sequence text to find the best match
with reference sequence◦ Robust alignment with DNA migration time: when two sequences are matched,
the DNA migration times slope of the two match sequence are generated. ◦ Maximum correlation for local adjustment
if insertion occur- a compression area will form and if a deletion occur the a gap will be formed so that bases from previous and after will still align with the reference sequence in order to illustrate the indels then the center line will turn red from green and a heavy line will draw above the point
Heterozygous indel detection◦ Using a method to de-convolute the heterozygous trace into two clean sets of
sequence traces according to the reference trace Use robust alignment to align good traces -> the same slope is generated for
the overlapping region of mixture (shown roughly align to reference trace)
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sample trace
Conserve trace identical to reference trace
mutation trace left after reference subtraction
Shift trace
Deletion detection
Mutation Surveyor and indel mutation detection
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Mutation Surveyor and heterozygous deletion case
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Heterozygous deletion case
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Mutation Surveyor and sensitivity
very sensitive for detection of rare variants which present as low as 10% of the population using the 2Dir smaller peaks
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Mutation Surveyor and analysis of multiple genes
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Mutation Surveyor and TTR mutation detection
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Developed by Interactive Biosoftware (IBS)-France
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ALAMUT : software and database
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ALAMUT: information for navigation
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How to select your gene of interest
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LDLR GENE
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Different sources of database can be linked
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SNPs viewing
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Different levels of search
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Protein multi-alignment
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Variant prediction tools
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Result interpretation
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Database links
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Database links
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ALAMUT : Add new mutation to database
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Analysis SCN1A mutations in a large number of samples using ◦ Seqcape v2.5 for sequence variant detection◦ Alamut v.131 software for
assessing mutation interpretation and amino acid conservation in orthologues
Use GD to test the effect of residue replacement with respect to a range of physicochemical properties (volume, charge and composition of side chain)
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The software was assessed in four areas◦ Its user interface and usability◦ The suitability of its data sources◦ Its applicability to diagnostic testing◦ Its validity and accuracy
The software was assessed by◦ National Genetic Reference Lab Manchester◦ Leiden University Medical Centre◦ GUY’S Hospital London◦ PRAGUE
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EuroGentest Unit 5 on decision support for molecular diagnostic laboratories using Interactive Biosoftware Alamut prepared by NGRL Manchester
http://leedsdna.info/HUGO/2004/Lab_Notes/2004_labs.htm