fourth quarter: atlas analysis 钟树荣( phd ,副教授) 昆明医科大学法医学院
TRANSCRIPT
![Page 1: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/1.jpg)
fourth quarter: atlas analysis
钟树荣( PhD ,副教授)
昆明医科大学法医学院
![Page 2: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/2.jpg)
AMEL
D3S1358TH01
TPOX
D2S1338
D19S433
FGA
D21S11
D18S51
CSF1PO
D16S539
D7S820
D13S317
D5S818
VWA
D8S1179
1 integrated analysis vs. 16 separate runs1 integrated analysis vs. 16 separate runs
Information is tied together with multiplex PCR and data analysis
AmpFlSTR® Identifiler™ (Applied Biosystems)
![Page 3: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/3.jpg)
![Page 4: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/4.jpg)
In STR analysis practice, not every batch of every sample analysis, can be completed accurately
and automatically by computer program
• Sometimes sample automatic classification result is wrong, need professional personnel adjustment parameter analysis, to analysis to obtain the correct classification
![Page 5: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/5.jpg)
Affect the classification
• 一、 Classification standard allelic naming errors
• 二、Within the logo don't mistake
• 三、 Base jumping or swing
• 四、 stutter peak
• 五、 A template depends on A
![Page 6: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/6.jpg)
Affect the classification
• 六、 Other pseudo peak
• 七、 Gender recognition graph anomalies
• 八、 Peak of the balance
• 九、 Heterozygosity lost or 3 alleles
• 十、 off-ladder peak
![Page 7: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/7.jpg)
一、 Classification standard allelic naming errors
Ladder, each each loci alleles are not completely equal amounts. After electrophoresis detection, the signal strength is different also
![Page 8: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/8.jpg)
If a loci of the a allele fragments: Volume is relatively small, the sample quantity is less,
and the peak threshold is set too high
All the alleles are reduce a repeating unit
![Page 9: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/9.jpg)
1、 If a particular loci alleles first fragment: Appear before small a repeating unit of pseudo peak (copy slip product), sample quantity is big,
and the peak on the threshold value is set too low
All the alleles increase a repeating unit name
![Page 10: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/10.jpg)
replication slippage : The main cause of
STR polymorphisms
![Page 11: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/11.jpg)
Action Taken
• 1、 Synchronous analysis of known reference samples ( 9947A)
• 2、 Check whether the loci alleles named Ladder is accurate.
![Page 12: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/12.jpg)
二、Within the logo don't mistake
Identify the correct internal standard
![Page 13: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/13.jpg)
1、 Signal is too low Adjust peak threshold
Increase in scalar electrophoresis again
solve
solve
![Page 14: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/14.jpg)
1、 Analysis of range error
![Page 15: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/15.jpg)
To adjust the scope of analysis
200 300
340
350 400
解决
![Page 16: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/16.jpg)
3.Electrophoresis data is not complete
Internal standard fragment is not all of them Collection, internal standard larger pieces missing
More than 400 bp fragment don't read Electrophoresis again
![Page 17: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/17.jpg)
cause
• 1、 Environmental temperature has significant effects on speed of electrophoresis
• 2、 Electrode buffer, capillary, etc. Use too much times, Electrophoresis resolution decreased
![Page 18: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/18.jpg)
三、 Base jumping or swing
• Base jumping or swing : Baseline is too high, more than peak threshold
• Countless fragment peaks
![Page 19: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/19.jpg)
![Page 20: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/20.jpg)
![Page 21: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/21.jpg)
cause
• 1、 Call matrix file error or are not suitable for analysis
• 2、 Peak signal is too strong, jamming signals could not be eliminate completely
![Page 22: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/22.jpg)
take measures
• Avoid blindly increase the sample amount:
• Peak signal is too strong Reduce the sample quantity After dilution products, electrophoresis again
• The ideal peak charts : 1000-3000
![Page 23: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/23.jpg)
四、 stutter peak
• In STR classification mapping, often before a target STR allele peak, there is little the position of a repeating unit a weak signal peak
![Page 24: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/24.jpg)
This extra peak is due to the PCR amplification, fragments amplified copy slip form, called the shadow zone (stutter
band) or shadow peak stutter (peak).
![Page 25: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/25.jpg)
• Stutter peak height or peak area should not exceed 15% of the purpose of allele peak
• Always in front of the objective allele peak small one repeating unit
• Within the same loci, long alleles product relatively short allele stutter
• Peak signal is too strong and homozygous genes, are more likely to stutter peak was observed
![Page 26: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/26.jpg)
take measures • Augment To reduce the amount of
DNA samples
• electrophoresis Reduce the sample quantity, reduces the peak signal strength
• Mention peak threshold
• Adopting peak signal filtering
• With higher thermal stability of DNA polymerase activity increases
Affect the mixed samples
![Page 27: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/27.jpg)
五、 A template depends on A
• Still have A kind of phenomenon, in the PCR process is dependence on amplification products of template to add A
• DNA polymerase can, in the end of the amplification products of 3 '- nonspecific to connect A adenosine A
A------------------
------------------A
![Page 28: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/28.jpg)
Through the primer design and the amplification cycle after the end of heat preservation, can make each fragment amplification products 3 '- are at the
end add A A 预变性 95 ,11min℃ 变性
95˚C,1min
60 ,℃
60min
延伸72˚C, 1min
退火59˚C, 1min
35
![Page 29: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/29.jpg)
• Each allele fragments of all amplified product bases of DNA molecules are exactly the same. Electrophoresis in single piece
![Page 30: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/30.jpg)
• If the achieved only half of A process, can make the same allele fragments amplification contains added A and not A, two DNA fragments, which is A base.
• The a allele two amplification product in electrophoresis, were identified as two peaks. Two peak close to, in the base into a peak, fork pointed part formed two peaks, referred to as a double peak.
![Page 31: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/31.jpg)
• Add A product peak fragment size and corresponding allelic ladder Genes in same size, was named digital
![Page 32: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/32.jpg)
• Without A product, no corresponding allelic ladder, was identified as off - ladder
![Page 33: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/33.jpg)
cause
• 1、When amplification DNA template
• 2、 Amplification system, with PCR inhibitor
• 3、 Taq polymerase or severe attenuation due to less quantity.
![Page 34: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/34.jpg)
attention
• 1、 Double peak phenomenon in small fragments alleles than large fragments, etc A gene is more common.
• 2、 If on a sample of data, in a larger pieces Fixed alleles in a single peak, then to consider Samples in the loci alleles micro variations may occur
![Page 35: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/35.jpg)
六、 Other pseudo peak
• In the electrophoresis process, when equipment appear short pulse output current, peak figure will appear in the data signal baseline suddenly beating.
• Peak in the figure is shown as figure is too high the fault type of peak suddenly, the pseudo peak may be identified as the fragment peaks
• "Wedge" peak
![Page 36: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/36.jpg)
cause
• Buffer containing solid particles, the particle surface if the negatively charged
• Stick the peak charts
• With d = 0.25 um filter, water filter for compound electrode buffer
![Page 37: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/37.jpg)
![Page 38: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/38.jpg)
七、 Gender recognition graph anomalies
• At present the commonly used commercial kits
• AmpFlSTR® Identifiler
• AmpFlSTR® SinofilerTM
• PowerPlex ® 16
Amelogenin(性别 STR 基因座)
“15+1”STRGene Locus
![Page 39: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/39.jpg)
• For most individuals : Amelogenin detection, can accurately identify the sex of the individual.
![Page 40: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/40.jpg)
Abnormal one
• Male individual Y chromosome AmelogeninRare is missing
• Don't have access to the Y chromosome amplification products
• Men samples were wrong that is female
• Y-STRamplification
expression
result
solve
![Page 41: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/41.jpg)
Exception 2
• Male individual X chromosome amelogenin Rare is missing
• Only Y chromosome amplification products
• Men's samples didn't detect the X chromosome
• X-chromosome Amelogenin Primer amplification
expression
result
solve
![Page 42: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/42.jpg)
八、 Peak of the balance
![Page 43: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/43.jpg)
• A loci of two segments of heterozygote peak signal strength differences of the phenomenon is more common, the main reason is that amplify imbalance
• Electric sample, small fragments faster into the capillary Small pieces was slightly higher than the peak peak larger pieces.
![Page 44: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/44.jpg)
![Page 45: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/45.jpg)
• Typically, small fragments peak area is bigger than big fragment peaks.
• However, peak area should be larger pieces in a small segment of the peak area More than 70%. That is the same different loci alleles The peak area of difference should be within 30%.
• If the two alleles of peak height difference is more than 70%, you will need to consider may be mixed samples
![Page 46: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/46.jpg)
cause
• 1、 Amplification DNA template is too little
• 2、 DNA Template degradation
• 3、 , exist in the PCR reaction system inhibitors: hemoglobin and its derivatives, etc
![Page 47: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/47.jpg)
Loss of heterozygosity
• 1、 Loss of heterozygosity
• Heterozygous samples in the corresponding loci detected one allele, similar to homozygous test results.
• But in peak area values and signal strength and other heterozygote heterozygote peak area of sample.
Tumor samples
![Page 48: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/48.jpg)
![Page 49: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/49.jpg)
九、 Third-class a gene
Cause:The extra chromosome; Primer combination point mutations
![Page 50: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/50.jpg)
• Samples should be at the check out 3 or multiple genes 4 allele
And pollution is more than often affected with batches of samples.
![Page 51: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/51.jpg)
十、 Off-Ladder 峰1
、LIZ
Identification
error
![Page 52: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/52.jpg)
To adjust the scope of analysis
200 300
340
350 400
solve
![Page 53: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/53.jpg)
2 、 Laddernaming errors
![Page 54: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/54.jpg)
3
、Mu
tant
allele
![Page 55: Fourth quarter: atlas analysis 钟树荣( PhD ,副教授) 昆明医科大学法医学院](https://reader035.vdocuments.site/reader035/viewer/2022081415/56649ed15503460f94be0775/html5/thumbnails/55.jpg)
The influencing factors of spectrum analysis
• sample
• electrophoresis
• external environment