Embed Size (px)
Citation preview
1/7/17
1
NGS Data Quality ControlSuzanne M. [email protected]
Copyrighted © S.M. Leal 2017
Phenotype Quality Control
• Select samples on one or more qualitative and or quantitative phenotypes
• For covariates missing values are replaced with average values for the cohort– e.g. Body mass index (BMI)
• Quantitative traits– Outliers
• windorize– If data violates normality
• QQ normalization
• Phenoman Li et al. 2014 Bioinformatics
NGS Data Quality Control
• Extremely important to perform before data analysis– Poor data quality can increase type I and II errors
• Sequence quality can be influenced by– DNA quality– Sequencing machines (read length)– Sequencing depth– Alignment – Variant Calling
• SNVs and Indels
NGS Data Quality Control (QC)
• Protocols for data control are in their infancy• No set protocols for QC• QC which has to be performed is data specific– Dependent on read depth – Batch effects– Availability of duplicate samples
NGS Data Quality – Removal of Genotype Calls
• Sequence read genotype depth (GD)• Concerned if GD is too low or too high*
– GD too low insufficient reads to call a variant site– GD too high can be an indication of copy number variants which can introduce false positive variant calls » *Due to down sampling in GATK maximum GD is 250
• Remove genotypes with low read depth, e.g. DP<8 • and high read depth e.g. >249
– Genotype quality (GQ) score• Removal of sites with low genotype quality core, e.g. GQ< 20
Removal of Individuals (Samples) due to Missing Data
• Remove individuals who are missing genotype calls/variant sites, e.g. > 10%– This is to remove individuals with bad quality data who can potentially have bad genotype calls
• If using different capture arrays need to make sure the intersect is used
1/7/17
2
NGS Data Quality ControlRemoval of Variant Sites
• Removal of sites with missing data– e.g. missing > 10% of genotypes
• Removal of “novel” variant sites which only occur in one batch and the alternative allele is observed multiple times or the minor allele frequency is high in overall sample
• Removal of sites that deviate from HWE– Must be performed by population if the study consists of more than one ethnic group, e.g. African American and European American
– Related individuals should also be removed from the sample
NGS Data Quality Control• Values which are used for GD, GQ and missing data cut offs are based upon – Concordance rates
• if there duplicate samples are available– Ti/Tv ratios
• For individuals• Entire sample
– Removal of batch effects • As evaluated by multidimensional scaling (MDS) or• Principal components analysis (PCA)
– Amount of data removed• Quality control can remove substantial amounts of data which should be avoided e.g. >15% of variant sites
NGS Data Quality Control• After Variant Quality Score Recalibration (VQSR) or– GATK
• Support Vector Machine (SVM)– UMAKE pipeline
• These routines are used to determine sites of bad quality
• However even after this step – concordance of duplicates (when available) and – and Ti/Tv ratios are often low
• Variant Association Tools (VAT)– Can be used for additional data cleaning
Transition/Transversion (Ti/TV) Ratios
A C
GT
TransitionTransversion
• Transition• Purine Purine• Pyrimidine Pyrimidine
• Transversion• Purine Pyrimidine• Pyrimidine Purine
Transition/Transversion (Ti/TV) Ratios
A C
GT
TransitionTransversion
• Ti/Tv Ratios• Whole genome ~2.0 • Exome novel ~2.7 • Exome known ~3.5
• Ti/Tv ratios can be calculated by • Sample or• Dataset
• Ti/Tv ratios can be evaluated for subsets of the data• e.g. by batch
QC-‐Pipeline
See Figure 2 in Wang et al. AJHG 2014
1/7/17
3
Example -‐Project Description• 1,667 Samples• Seven cohorts• Two sequencing centers– Center 1
• Two capture arrays– NimbleGen V2Refseq 2010 (CA1): 1082
» Batch 1 and 3– NimbleGen bigexome 2011 (CA2): 234
» Batch 2– Center 2
• One capture array– Agilent SureSelect
» Batch 4
• Four batches• No intentional duplicate samples
Example Project Description• Intersection of the three capture arrays used– NimbleGen V2Refseq 2010
• Batch 1 and 3– NimbleGen bigexome 2011
• Batch 2– Agilent Sure Select
• Batch 4• Sequencing machine– Illumina HiSeq
• Sequence alignment– BWA
• Multi-‐sample variant calling– GATK
Sequence Data QC Overview
• Variant and genotype call level– Evaluation of batch effects
• Genotype call level – Removal of genotype calls– Low or high depth of coverage DP < 8 & DP > 249– Low genotype quality score GQ < 20
Sequence Data QC Overview
• Removal of individual samples– >20% missing data
• After taking the intersect of capture arrays– Samples without phenotype information
• Variant level – removal of variant sites– Low call rate
• i.e., missing call rate > 10%– “Novel” variant sites observed >2 only in a single batch– Deviation from Hardy-‐Weinberg-‐Equilibrium
• Population specific• Unrelated individuals
– p<5 x 10-‐7
Sequence Data QC Overview
• Detection of sample outliers – Perform multidimensional scaling (MDS) to detect outliers• Due to population substructure/admixture and batch effects• Remove effects by
– Additional QC– Removal of outliers and\or – Inclusion of MDS or PCA components in the association analysis
Sequence Data QC Overview• Evaluate sex of individuals based upon X and Y
chromosomal data– Sample mix-‐ups– Individuals with Turner or Klinefelter Syndrome
• Evaluate samples for cryptically related individuals and duplicate samples– King or Plink algorithm– Retain one duplicate of a pair– Retain only individual of a relative group or control for relatedness in the analysis
• Post Analysis -‐ Quantile-‐Quantile (QQ)plots– To evaluate uncontrolled batch effects and population substructure/admixture
1/7/17
4
Evaluating Batch Effects
Mean DP by Batch
Mean GQ by Batch Genotypes Removed by DP Cut-‐offBy Batch
●●
●●
●●
●●
●●
●
●
●
●
0%
5%
10%
15%
20%
25%
30%
35%
40%
1 2 3 4 5 6 7 8 910 15 20 30 40Genotype Depth Cutoff
Gen
otyp
es R
emov
ed
Batch● Batch1
Batch2Batch3Batch4
Genotypes Removed by DP Cut-‐offBy Batch (GQ >20)
● ● ● ● ● ● ● ● ●●
●
●
●
●
0%
5%
10%
15%
20%
25%
30%
35%
40%
1 2 3 4 5 6 7 8 910 15 20 30 40Genotype Depth Cutoff
Gen
otyp
es R
emov
ed
Batch● Batch1
Batch2Batch3Batch4
Genotypes Removed by GQ Cut-‐offsBy Batch
●
●
●
●
●
●
●
●
●
●
0%
5%
10%
15%
20%
0 5 10 15 20 25 30 35 40 45Genotype Quality Cutoff
Gen
otyp
es R
emov
ed
Batch● Batch1
Batch2Batch3Batch4
1/7/17
5
Genotypes Removed by GQ Cut-‐offsBy Batch (DP>8)
● ● ● ● ●
●
●
●
●
●
0%
5%
10%
15%
0 5 10 15 20 25 30 35 40 45Genotype Quality Cutoff
Gen
otyp
es R
emov
ed
Batch● Batch1
Batch2Batch3Batch4
Missing Rate Criteria & Sites Removed
10% 5%
Before QC* 2.5% 3.9%
After QC 12.9% 18.3%
Variant sites missing >10% of their data were removed
*After VQSR
Ti/Tv Ratios during QC Process
Known Novel All
Before VQSR 2.95 ± 0.05 1.18 ± 0.29 2.86 ± 0.07
Before QC 3.12 ± 0.03 2.01± 0.32 3.11± 0.03
Genotype QC DP<8, GQ <20 3.18± 0.04 2.10±0.32 3.16± 0.03
Remove sites missing >10% genotypes 3.39± 0.04 2.42± 0.52 3.39± 0.04
Remove batch specific novel sites > 2 N=17,835 3.39± 0.04 2.41± 0.53 3.39± 0.04
Remove sites deviating from HWE p<5x10-‐7N=4,414 3.41± 0.04 2.39± 0.54 3.40± 0.04
Ti/Tv Ratios by IndividualBefore and After QC
All Known Novel All Known Novel Before QC After QC
Ti/Tv Ratios
Detecting Outliers Using Multidimensional Scaling (MDS)
• Multidimensional Scaling (MDS) and principal components analysis (PCA) are frequently used to detect outliers – MDS & PCA can also be used to control for substructure in the analysis
• Outliers can be cause by – Population stratification– Population substructure– Batch Effects
Sequence Data QC
• Batch effects can sometimes be removed with additional QC
• Extreme outliers should be removed• Additionally MDS or PCA components can be included in the analysis to control for population substructure\admixture and batch effects– Unless correlated with the outcome (phenotype)
• Or for batch effects (dummy coding) may be included as a covariate in the analysis– Unless correlated with the outcome (phenotype)
1/7/17
6
MDS First 2 Components Before QC*
●
●●
●
●
●
●●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●
●●
●
●
●
●
●
●
●
●
●
●
●
●●●
●
●
●
●
● ●
●
●●
●●
●
●
●
●
●●
●
●●●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
● ●●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
● ●
●
● ●●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
● ●
●
●●
●
●
●●
●
●
● ●● ●
●●
●
●
●
●
●
●●●
●
●
●
●
●
●
●
●
●
●
●
● ●
●
●
●
●
●
●●
●●
●
● ● ●●
●
●
●
●
●●
●
●●
●
●● ●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
● ●●
●
● ●
●
●●
●
● ●
●●
●●
●
●
●
●● ●
●
● ●
●
●
●
●
●
●
●
●
●
●
● ●
●●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
● ●●
●
●
●
●
●●
●●
●●
●
●
●
●
●
●
●
●
●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●●●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●●
●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
● ●
●●●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●● ●●
●
●
●●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●●
●●
●
●
●
●
●
●●
●●
●
●
●
●
●
●●●
●
●
●
●●
●
●
●
●●
●
●
●
●
●
●
●
● ●
●
● ●
●●
● ●
●
●
●
●●●
●
●●●
●
●
●
●
●●
● ●
●
●
●
●
●
●●
●
●●
●
●
●●●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●●
●
● ●
●
●
●
●
●
●
●●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
● ●
●
●●
●
●
●●
●
●
●
●
●
●
●●
●
●
●
●
●● ●●
●
●●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●●
●●
●
●
●
●
●
●●
●●
●
●
●
●● ●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
● ●
●
●
●
●
●
●
●
●
●
● ●
●
●
●●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●● ●
●
●●
●
●●
●●
●
●
●
●
●
●●
●
●
●
●
●●
●
●●
●
●
●
●
● ●
●
●●
●
● ●●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
● ●●
●
●
●
●
●
●●
●
●
●
●
●
●●●
●
●
●●
●
●
●
●
●
●
●●
●
●
●
●
●●●
●●
●
●
●
●
●
●●
●
●
● ●
●
●
●
●
●
●
●
●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
● ●
●●
● ●●
●
●
●
●
●
●
●●
●●
●
●
●
●
●
●
●
●●
●●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●●
●
●
●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●●
●●
●●
●
● ●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
● ●
●
●
●
●●
●
●
●
●●
●
●
●
●●●
●
●
●
●
●●
●
●
●
●
●●●●
●
●●
●
●
●
●●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
● ●
●
● ●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●●
●
●
●
●
●●
●●
●
●
●
●
● ●● ●
●●
● ●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●●
●●
●
●
●●
●
●●
●
●
●
●
●
●●●
●
●
●
●
●
●
●
●
●
●
●
●●
●● ●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●●
● ●
●
●● ●
●●
●
●●
●
●
●
●●
●
●
●●
●
●●
●
●●
●●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●
●●
●●
● ●
●●
●
●●●●●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●●
●●
●●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●● ●●
●
●
●
●
●●
●●
●
●●
●
●
●
●
●
●●
●
●
●●●●
●
●●
●
●
●
●
●
●
●
●
●
●●
●
●
●●
●
●
●●
●●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●
●
●
●
● ●●
●
●●
●
●
●
●
●●
●
●
●●● ●
● ●●
●
●
●
●
●●●
●●
●
●
●●
●
●
● ●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●●
●
●
●
●
●
●
●●●
●
●
●●
●
●
●●
● ●
●●
●
●
●
●●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●●
●
●
●
●
●
●
●●
●
●●●
●
●●
●
●
●
● ●
●
●●
●
●
●
●●●
●
●
●
●
●●
●
●● ●
●●
●
●●
●
●
●●●
●
●●
●
●
●
●
●
●
●●
●
●
●●
●●
●
●
●
●
●
●
●●
●
●
●
●●
●●
●●
●
●
●
●
●
●
●●
●
●
●
●
●●
●
●
●
●
●
● ●
●
●
●● ●
●
●
●
●
●
●
●
−0.050
−0.025
0.000
0.025
−0.05 0.00 0.05 0.10 0.15 0.20
king_all_variant_MDS1
king_all_variant_M
DS2
batch●
●
●
●
1234
*After VQSR
●●●●●●
●
●●●
●● ●● ●●●
●●●
●●
●
●● ●
●●
●
●
●
●●
●
● ●●
●
●●●
●●
●
●
●●●
●●
●
●●
●
●●
●●●●● ●
●
●●
●●
●
●
●
●
● ●●
● ●●●
●
●●
●●
●
●
●●
●●●
●●
●●
● ●
●
●
●
●● ●●●
● ●
●●●●● ●
●
●
●
●● ●●
●●
●
●
●
●●
●
●
●
●●
●
●●
●●
●
●
● ●
●
●●
●●●
●
●●
●●
●●
●●
●●
●
●
●●
●
●●
●●
● ●● ●●
●
●● ●
●●●
● ●
●
●●
●●
● ●
●
●
●
●● ●
●●
●
●●
●
●
●
●●●
●
●●●
●
●
●
●●
●●
● ●●
●●
●●● ●
● ●●●●
●●
●●
●
●
●●
●
●
●●●
●
●
●
●● ●●●
●
●
● ●● ●
●●
●● ●● ●●●
●●●
●●●
●●
●
●
●●●●●
●
●●●
●
● ●●
●●
●
●●● ●
●●
●
● ●●
●●
● ●● ●
●●●●
●
●
●●
●●
●● ●●
●
●●
● ●●
●
●
●●●
●● ●●
●●●
●
●●
●
●●●
●
●●
●●●●●
●●
●● ●
●● ●
●
●●
●
●●
●●
●●
●●
●
●
●
●
●●
●
●
●
●●●
●●
●
●●
● ●
●●
●
●●
●●
●●
●
●
●
●
●●●
●
●●
●
●●
●● ●●
●●
● ●
●
●●
●
●●
●
●
●
●●
●●
●●●
●●
●●●
●
●
●
●●
●●
●● ●●
●
●
●
●●●
●●
●
●
●
●
●
●●
●
●●
●
●
●
●
●
●● ●● ●● ●●
●●
●
●●
● ●●
●
●
●
●
●●
●
● ●●
●●
●
● ●
●●
●●●
●●
●●
●●
●
●●●
●●●
● ● ●
●●
●
● ●●
● ●
●
●
●
●●
●
●
● ●●●
●●
●●●●●●
●●
●●
●●
●●
●●
●
●● ●●
●●
●●
● ●●
●
●
●
●●●
●● ●
●
●●●
●●
●
● ●
● ●
● ●●
●
●●
●
●
●●● ●
●
● ●●●
●
●●
●
●●●● ●
●●
●
●●
●
● ●● ●●●
●●●
●
●
●
●
●
●
●●
●●●
●●
●
● ●●
●● ●
●●
● ●
●
●
●●●● ●●
●●
●●●●
●
●
● ●●●● ●
●●●
●
● ●●●●
●
●●
●●
● ●● ●●●
●
●
●
●
●
●
●● ●
● ●
●● ● ●
●
●●●
●
●●
●●●
●
●
● ●
●●
●
● ●
●
●
●
● ●●●
●●
●●
●
●●
●
●●
●
● ● ●●
●●●
●●
●●
●
● ●●
●
●●
●
●
●●
●●●●
●●
●
●● ●
●●
●
●
●●●●●
●
●●●
●
●
●
●●
●
●●
●
●
●●
●●
● ●
●●● ●
●
●●
●●
●●
●
●
●● ●●
●● ●
● ●●
●●
●
●
●
●
●●
●
●●●
●
●
● ●●
●
●●● ●
●
●
●●●
●
●●●●
● ●●● ●
● ●
●
●● ●
●●
● ●
●
●
●
●
●
●●●
●
●
●●
●
● ●●●
●
●
●● ●
●
●
●● ●
●●
●●●●●
●
●●
●●
● ●●
●
●●
● ●●●
●●●
●●
●
●
●●●● ●
● ●
●●
●
●
●
●
●●
●
●●●
●●
●
●●
●●
●●
●●
●●
●
●
●
●
●
●●
●
●●
●
● ●●●●
●
●
●●
●
●
●
●
●
●
●●
●
●●
●●
●
●●
●
●
●● ●
● ●
●
●●● ●
●●●● ●
●●
●●
●●●
●●
●●
●
●
●●
●●●●●
●●
●
●●●● ●
●●
●
●
●●
● ●● ●●
●●●
●
●● ●
●
●
● ●● ●
●
●
●
●
●
●
●●
●●● ●
●●
● ●●
●
●
●●
●
●
●●
●●
●
● ●●
●
●
●
●
●
●
● ●●
●
●●
●
●●
●
●●
●
●
●●
●
●
●
●
●●●
●●
●
●
● ●●
●●●
●
● ●●●
● ●
●
● ●●
●
●●
●
●
●
●● ●
●●
●●●
●
●
●
●
●●
●
●●
●
●●
●
●
●
●●
●●● ●
●●●●
●
●●●
●●
●
●●
●●
●
●●
●
●●
●
●
●
●
●
● ●
●
●
●●
●●
● ●
●
●●●
●●
●●●
● ●
●●
● ● ●●●
●●
●●
●
●●●
●●
●
●●●
●●
● ●●
●●
●●
●●● ●
●
● ●●
●●
●
●●
●
●● ● ●
●
●
●● ● ●●●
●
●
● ● ● ●●
●
●●
● ●
●
●
●
●
●●
●
●
● ●
●●
●●●
● ●●
●
●
●
●●
●●
●
●
●●
●
●●
●●●
●●
●
●
● ●
●● ●
●
●●●● ●
●●
●●●
● ●
●●
●●
●
●
●●● ●●
●●
●●
●●
●
●
●
●
●●●●
●
● ●●
●
● ●●
●
●●● ●
●
●● ●
●
●●
●●
●
●●●
●
●●
● ●●
●●
●● ●●
●●
●●●
●●
●●● ●●
●
●●
●● ●
●
●
●
●●
●●●
●
●
●●●
●●
●
●●
●●
●●●
● ● ●
●
●●
●
●
●●
●●
●
●●●
●●
●● ●●
●
●●
●●
●
●
●
● ●●●
●●
●
●
● ●●● ●
●
●
● ●
●
●● ●● ●●
●●
●
●
●
●●
●
●● ●●
●●
●
●
●●●
●
●
●●
●
●
●● ●● ● ●
●●●●●
●●
●●●
●●●
●●
●
●
●
●●
●●
●
●
●
●
●● ●
●
●
●
●●●●
●●● ●
●
●
●●
●●
●
● ●
●
●●
●●0.0
0.1
0.2
0.3
0.4
0.5
−0.05 0.00 0.05 0.10 0.15 0.20
king_VLQC_BMR10_MDS1
king_VLQ
C_BMR10_M
DS2
batch●
●
●
●
1234
MDS First 2 Components After QC
Samples excluded
Reason NumberMissing primary phenotype 26
Excluded due to not meeting phenotype criteria 118
High genotype missing rate (> 20%) 11
1st and 2nd MDS components plot outliers 6
Total 176
Total number of samples 1506 for analysis From a total of 1667
Checking for Cryptic Related Individuals and Duplicate Samples
• Inclusion of related individuals/duplicate samples can increase type I and II errors
• Only one individual for a relative group or one individual from a duplicate sample should be retained in the analysis– Based upon availability of phenotype data and quality of sequence data
Checking for Cryptically Related Individuals and Duplicate Samples
• If related individuals are included in the analysis then to control type I and II errors– Mixed models should be used to analyze the data or – p-‐values can be obtained empirical– Fixed effects model including MDS or PCA components is not sufficient
Checking for Cryptically Related Individuals and Duplicate Samples
• PLINK algorithm which is based on identical-‐by-‐descent (IBD) statistic under the assumption of homogeneous population or
• KING-‐robust algorithm which uses an estimate of the genome-‐wide average heterozygosityacross individuals to compute an estimator of kinship coefficient
• VAT implements the KING-‐robust algorithm
1/7/17
7
Checking for Cryptically Related Individuals and Duplicate Samples
• PLINK algorithm– Can cause unrelated individuals to look related when there are batch effects or population substructure/admixture
• KING-‐robust algorithm– Problem is not as severe
Cryptic Relatedness
Analysis all Samples Analysis by Batch
Cryptic Relatedness Third Degree Relatives
Analysis all Samples Analysis by Batch
Related individuals
Type Pairs IndividualsMZ twin\or Duplicate 1 2
1st degree 50 78
2nd degree 42 68
3rd degree 97 101
Total 190 209
Evaluating Data QC Post-‐AnalysisQuantile-‐Quantile Plots
• Plot – –ln (observed p-‐values) vs –ln (expected p-‐values)
• Deflated p-‐values (smaller than expected)– Observed as points above the horizontal line• Indicating type I errors
• Inflation of the p-‐values (larger than expected)– Observed as points below the horizontal line• Indicating type II errors
Evaluating Data QC Post-‐AnalysisQuantile-‐Quantile Plots
• Lambda which is the standardized mean of the test statistic is used evaluated if there is inflation λ<1 or deflation λ>1 of the p-‐values– However caution should be used – there can still be deflation of the p-‐values (increased type I error) when λ=1
1/7/17
8
QQ Plots
• Inflation of p-‐values (type II errors) when the – Study is underpowered
• Deflation of p-‐values (type I errors) when there is– Population substructure/admixture– Batch effects– Different missing rates of genotype data between cases and controls• Only true for aggregate rare variant association tests
– Related Individuals– For quantitative traits
• Deviations from normality
Quantile-‐Quantile (Q-‐Q) PlotRare Variant Association Analysis
●
●
●●
●●
●●●
●●●●●
●●●●●●●
●●●●●●
●●●●●●●●●●●●●
●●●●●●●●●●●●●●●●●●●
●●●●●●●●●●●●●●●●●●●
●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●
●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●
●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●
●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●
ISY1
ZNF436
0
2
4
0 1 2 3 4− log10 pexpected
−lo
g 10 p
obse
rved
cmc method
![NGS 16 Series / 표시형식 NGS 20 Series NGS[H]-F 16 RD 6-T-1-J 1 1 · 2017-12-08 · Impeller (NGS 16 / NGS-F16) 고효율과 내구성을 추구한 독자적 설계. Compact Magnetic](https://img.pdfslide.tips/doc/110x75/5f880f271462972dab565017/ngs-16-series-oeoe-ngs-20-series-ngsh-f-16-rd-6-t-1-j-1-1-2017-12-08.jpg)
