Huong LeDepartment of Molecular & Clinical

Genetics,Royal Prince Alfred Hospital

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Sequence variant detection SOFTWARES

Sequence variant interpretation SOFTWARE

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

Seqscape and cDNA annotation set up

Seqscape and cDNA annotation set up

Seqscape – Genotype Table

c. 681 location

g. 2479065 location

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

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

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

Mutation Surveyor and result output

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)

sample trace

Conserve trace identical to reference trace

mutation trace left after reference subtraction

Shift trace

Deletion detection

Mutation Surveyor and indel mutation detection

Mutation Surveyor and heterozygous deletion case

Heterozygous deletion case

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

Mutation Surveyor and analysis of multiple genes

Mutation Surveyor and TTR mutation detection

Developed by Interactive Biosoftware (IBS)-France

ALAMUT : software and database

ALAMUT: information for navigation

How to select your gene of interest

LDLR GENE

Different sources of database can be linked

SNPs viewing

Different levels of search

Protein multi-alignment

Variant prediction tools

Result interpretation

Database links

Database links

ALAMUT : Add new mutation to database

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)

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

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