Try different quantification methods

Last updated: 2020-03-23

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Knit directory: Comparative_APA/analysis/

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Unstaged changes:
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Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.

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File	Version	Author	Date	Message
Rmd	0e55318	brimittleman	2020-03-23	diagnose multimap
html	02f5c26	brimittleman	2020-03-20	Build site.
Rmd	a4d172b	brimittleman	2020-03-20	add mm res
html	246d60c	brimittleman	2020-03-19	Build site.
Rmd	b6ccdbc	brimittleman	2020-03-19	add new quat

library(tidyverse)

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library(ggpubr)

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library(Matrix)


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I am worried about using featureCounts. It looks like there are reads that are not counted. In this analysis, I will look at correlation between counts as well as the specific locations I have been worried about.

First, I will do this in human nuclear with the final PAS.

This is the fixed strand. The

Meta=read.table("../data/PAS_doubleFilter/PAS_10perc_either_HumanCoord_BothUsage_meta_doubleFilter.txt",header = T)
ChimpMeta=read.table("../data/PAS_doubleFilter/PAS_doublefilter_either_ChimpCoordChimpUsage.sort.bed", col.names = c("chr","start","end", "PAS", "score", "strand"),stringsAsFactors = F) %>% select(PAS)
HumanCountsFeatureCounts=read.table("../Human/data/CleanLiftedPeaks_FC/ALLPAS_postLift_LocParsed_Human_fixed.fc", header = T) %>%  separate(Geneid, into=c("disc", "PAS", "chr2", "start2", "end2", 'strand', 'geneid'), sep=":")  %>% select(-contains("_T")) %>% filter(PAS %in% Meta$PAS) 

ChimpCountsFeatureCounts=read.table("../Chimp/data/CleanLiftedPeaks_FC/ALLPAS_postLift_LocParsed_Chimp_fixed.fc", header = T) %>%  separate(Geneid, into=c("disc", "PAS", "chr2", "start2", "end2", 'strand', 'geneid'), sep=":")  %>% select(-contains("_T")) %>% right_join(ChimpMeta,by="PAS")

First use the bedtools method:

mkdir ../data/testQuant

sbatch humanMultiCov.sh

sbatch chimpMultiCov.sh

HumanDFMulticov=read.table("../data/testQuant/Human_DF_PAS.txt")

Compare:

# cor(a,b)

HumanCountsFeatureCounts_mat= as.matrix(HumanCountsFeatureCounts %>% select(contains("_N")))

HumanDFMulticov_mat=as.matrix(HumanDFMulticov[,7:12])
#PAS correlation 
Human_DFcor= cor(HumanCountsFeatureCounts_mat,HumanDFMulticov_mat)

I care about the diagonal for the matrix.

Human_DFcordiag= diag(Human_DFcor)

Human_DFcordiag

[1] 0.9839297 0.9551134 0.9668683 0.9742895 0.9781832 0.9821528

There are the correlations for individual.

ChimpDFMulticov=read.table("../data/testQuant/Chimp_DF_PAS.txt")

ChimpCountsFeatureCounts_mat= as.matrix(ChimpCountsFeatureCounts %>% select(contains("_N")))

ChimpDFMulticov_mat=as.matrix(ChimpDFMulticov[,7:12])
#PAS correlation 
Chimp_DFcor= cor(ChimpCountsFeatureCounts_mat,ChimpDFMulticov_mat)

Chimp_DFcordiag= diag(Chimp_DFcor)

Chimp_DFcordiag

[1] 0.9900335 0.9727290 0.9819526 0.9805695 0.9751067 0.9772205

Compare species

DF_Multi=as.data.frame(cbind(Chimp=Chimp_DFcordiag,Human=Human_DFcordiag)) %>% gather("Species", "Correlation")


ggplot(DF_Multi,aes(x=Species, y=Correlation, fill=Species)) + geom_boxplot() + geom_jitter() + stat_compare_means() + labs(title="Double filtered Final PAS correlation \nbetween feature counts and multiCov")

Version	Author	Date
246d60c	brimittleman	2020-03-19

Difference in the matricies:

HumanDiff= HumanCountsFeatureCounts_mat-HumanDFMulticov_mat
ChimpDiff=ChimpCountsFeatureCounts_mat-ChimpDFMulticov_mat

Extract the non

nnzero(HumanDiff)

[1] 110127

sum(HumanDiff)

[1] -1787591

HumanDiff_g=as.data.frame(HumanDiff) %>% gather("Ind", "count")

ggplot(HumanDiff_g, aes(x=Ind, y=count,col=Ind))+ geom_boxplot() + labs(y="FeatureCount - MultiCov", title="Human Difference in Counts for final PAS")

Version	Author	Date
02f5c26	brimittleman	2020-03-20

Look at the examples with the min values.

HumanDiff_rowS= rowSums(HumanDiff)
#min:  
minDiff=which(HumanDiff_rowS==min(HumanDiff_rowS))

#36964 (look at this row in the df)
HumanCountsFeatureCounts[minDiff,]

      disc         PAS chr2  start2    end2 strand    geneid  Chr   Start
36964 Both human299134 chr7 5527138 5527338      + ACTB_utr3 chr7 5527138
          End Strand Length X18498_N X18499_N X18502_N X18504_N X18510_N
36964 5527338      +    201     3833     4635     3328     2983     3460
      X18523_N
36964     4132

HumanDFMulticov[minDiff,]

        V1      V2      V3          V4        V5 V6    V7    V8    V9
36964 chr7 5527138 5527338 human299134 0.9833333  - 36243 45135 31796
        V10   V11   V12
36964 32458 33604 39187

nnzero(ChimpDiff)

[1] 112291

sum(ChimpDiff)

[1] -1904816

ChimpDiff_g=as.data.frame(ChimpDiff) %>% gather("Ind", "count")

ggplot(ChimpDiff_g, aes(x=Ind, y=count,col=Ind))+ geom_boxplot() + labs(y="FeatureCount - MultiCov", title="Chimp Difference in Counts for final PAS")

Version	Author	Date
02f5c26	brimittleman	2020-03-20

Look at the examples with the min values.

ChimpDiff_rowS= rowSums(ChimpDiff)
#min:  
minDiffChimp=which(ChimpDiff_rowS==min(ChimpDiff_rowS))

ChimpCountsFeatureCounts[minDiffChimp,]

      disc         PAS chr2  start2    end2 strand    geneid  Chr   Start
36910 Both human299134 chr7 5527138 5527338      + ACTB_utr3 chr7 5663709
          End Strand Length X18358_N X3622_N X3659_N X4973_N pt30_N pt91_N
36910 5663909      +    201     3626    7146    5153    6572   8693   7306

ChimpDFMulticov[minDiffChimp,]

        V1      V2      V3          V4    V5 V6    V7    V8    V9   V10
36910 chr7 5663709 5663909 human299134 0.985  - 31910 63880 44470 49514
        V11   V12
36910 73935 60607

This shows that in general counts are lower in the features counts.

Examples:

human188040

Meta %>% filter(PAS=="human188040")

          PAS  disc  gene loc  chr     start       end Chimp Human
1 human188040 Human PLCL1 cds chr2 198084933 198085133 0.095 0.025
  strandFix
1         +

ChimpDFMulticov %>% filter(V4=="human188040")

     V1       V2       V3          V4    V5 V6 V7 V8 V9 V10 V11 V12
1 chr2B 84660923 84661123 human188040 0.095  +  0  0  5   1   0   0

ChimpCountsFeatureCounts %>% filter(PAS=="human188040")

   disc         PAS chr2    start2      end2 strand    geneid   Chr
1 Human human188040 chr2 198084933 198085133      - PLCL1_cds chr2B
     Start      End Strand Length X18358_N X3622_N X3659_N X4973_N pt30_N
1 84660923 84661123      -    201        0       0       5       1      0
  pt91_N
1      0

HumanDFMulticov %>% filter(V4=="human188040")

    V1        V2        V3          V4    V5 V6 V7 V8 V9 V10 V11 V12
1 chr2 198084933 198085133 human188040 0.025  +  0  2  6   3   0   2

HumanCountsFeatureCounts %>% filter(PAS=="human188040")

   disc         PAS chr2    start2      end2 strand    geneid  Chr
1 Human human188040 chr2 198084933 198085133      - PLCL1_cds chr2
      Start       End Strand Length X18498_N X18499_N X18502_N X18504_N
1 198084933 198085133      -    201        0        2        6        3
  X18510_N X18523_N
1        0        2

Same counts here. This is why it was not found in chimp. It seems it wouldnt pass the non zero filter.
chimp171173

Meta %>% filter(PAS=="chimp171173")

          PAS  disc gene  loc  chr   start     end     Chimp        Human
1 chimp171173 Chimp ADI1 utr3 chr2 3498436 3498639 0.5358333 0.0008333333
  strandFix
1         -

HumanDFMulticov %>% filter(V4=="chimp171173")

    V1      V2      V3          V4           V5 V6  V7 V8  V9 V10 V11 V12
1 chr2 3498436 3498639 chimp171173 0.0008333333  - 140 87 105  47 250 525

HumanCountsFeatureCounts %>% filter(PAS=="chimp171173")

   disc         PAS chr2  start2    end2 strand    geneid  Chr   Start
1 Chimp chimp171173 chr2 3498436 3498639      + ADI1_utr3 chr2 3498436
      End Strand Length X18498_N X18499_N X18502_N X18504_N X18510_N
1 3498639      +    204        0        0        0        0        0
  X18523_N
1        0

chimp171172

Meta %>% filter(PAS=="chimp171172")

          PAS  disc gene  loc  chr   start     end Chimp Human strandFix
1 chimp171172 Chimp ADI1 utr3 chr2 3497907 3498106 0.155     0         -

HumanDFMulticov %>% filter(V4=="chimp171172")

    V1      V2      V3          V4 V5 V6 V7 V8 V9 V10 V11 V12
1 chr2 3497907 3498106 chimp171172  0  - 24  9 10  15  27 110

HumanCountsFeatureCounts %>% filter(PAS=="chimp171172")

   disc         PAS chr2  start2    end2 strand    geneid  Chr   Start
1 Chimp chimp171172 chr2 3497907 3498106      + ADI1_utr3 chr2 3497907
      End Strand Length X18498_N X18499_N X18502_N X18504_N X18510_N
1 3498106      +    200        0        0        0        0        0
  X18523_N
1        0

This is fixed. (ADI1)

LAT human124355

Meta %>% filter(PAS=="human124355")

          PAS  disc gene  loc   chr    start      end     Chimp Human
1 human124355 Human  LAT utr3 chr16 28990265 28990465 0.4241667  0.52
  strandFix
1         +

ChimpDFMulticov %>% filter(V4=="human124355")

                    V1     V2     V3          V4        V5 V6 V7 V8 V9 V10
1 chrUn_NW_019937196v1 380529 380729 human124355 0.4241667  - 19 26 43  23
  V11 V12
1  34  25

ChimpCountsFeatureCounts %>% filter(PAS=="human124355")

   disc         PAS  chr2   start2     end2 strand   geneid
1 Human human124355 chr16 28990265 28990465      - LAT_utr3
                   Chr  Start    End Strand Length X18358_N X3622_N
1 chrUn_NW_019937196v1 380529 380729      +    201       19      23
  X3659_N X4973_N pt30_N pt91_N
1      43      23     32     24

HumanDFMulticov %>% filter(V4=="human124355")

     V1       V2       V3          V4   V5 V6 V7 V8 V9 V10 V11 V12
1 chr16 28990265 28990465 human124355 0.52  + 50 10 28  18  33  32

HumanCountsFeatureCounts %>% filter(PAS=="human124355")

   disc         PAS  chr2   start2     end2 strand   geneid   Chr    Start
1 Human human124355 chr16 28990265 28990465      - LAT_utr3 chr16 28990265
       End Strand Length X18498_N X18499_N X18502_N X18504_N X18510_N
1 28990465      -    201       50       10       28       16       32
  X18523_N
1       31

Makes sense. Discovered only in human because on contig. Counting is ok.

Compare the PAS discovered in human:

humanDisc=Meta %>% filter(disc=="Human")

ChimpFC_human= ChimpCountsFeatureCounts %>% filter(PAS %in% humanDisc$PAS)
ChimpMC_human= ChimpDFMulticov %>% filter(V4 %in% humanDisc$PAS)

#correlation  
ChimpFC_human_mat= as.matrix(ChimpFC_human[,13:18])
ChimpMC_human_mat=as.matrix(ChimpMC_human[,7:12])
#PAS correlation 
Chimp_DFcor_Humandisc= cor(ChimpFC_human_mat,ChimpMC_human_mat)

Chimp_DFcor_Humandiscdiag= diag(Chimp_DFcor_Humandisc)

Chimp_DFcor_Humandiscdiag

[1] 0.9994462 0.9949149 0.9990672 0.9974558 0.9984791 0.9988820

Is the multicov higher?

ChimpHumanDiscDiff= ChimpFC_human_mat-ChimpMC_human_mat

nnzero(ChimpHumanDiscDiff)

[1] 12166

sum(ChimpHumanDiscDiff)

[1] -212818

summary(ChimpHumanDiscDiff)

    X18358_N            X3622_N             X3659_N         
 Min.   :-2248.000   Min.   :-2444.000   Min.   :-1658.000  
 1st Qu.:   -1.000   1st Qu.:   -2.000   1st Qu.:   -1.000  
 Median :    0.000   Median :    0.000   Median :    0.000  
 Mean   :   -8.554   Mean   :   -8.259   Mean   :   -6.238  
 3rd Qu.:    0.000   3rd Qu.:    0.000   3rd Qu.:    0.000  
 Max.   :  345.000   Max.   :  198.000   Max.   :  213.000  
    X4973_N             pt30_N              pt91_N         
 Min.   :-2526.00   Min.   :-2935.000   Min.   :-2713.000  
 1st Qu.:   -2.00   1st Qu.:   -2.000   1st Qu.:   -2.000  
 Median :    0.00   Median :    0.000   Median :    0.000  
 Mean   :   -6.81   Mean   :   -8.015   Mean   :   -8.682  
 3rd Qu.:    0.00   3rd Qu.:    0.000   3rd Qu.:    0.000  
 Max.   :  192.00   Max.   :  134.000   Max.   :  119.000

ChimpHumanDiscDiff_rowS= rowSums(ChimpHumanDiscDiff)
#min:  
minDiffChimpinH=which(ChimpHumanDiscDiff_rowS==min(ChimpHumanDiscDiff_rowS))

ChimpFC_human[minDiffChimpinH,]

     disc       PAS chr2   start2     end2 strand    geneid  Chr    Start
113 Human human8304 chr1 44778707 44778907      - RPS8_utr3 chr1 43588450
         End Strand Length X18358_N X3622_N X3659_N X4973_N pt30_N pt91_N
113 43588650      -    201      400     421     239     448    627    602

ChimpMC_human[minDiffChimpinH,]

      V1       V2       V3        V4  V5 V6   V7   V8   V9  V10  V11  V12
113 chr1 43588450 43588650 human8304 0.5  + 2648 2534 1757 2974 3562 3315

chr 1 (.5X) RPS8 (snoRNAs near- could be problem )

Redo with different map quality

-q 255 this will not count multimapping reads.

sbatch chimpMultiCov255.sh
sbatch humanMultiCov255.sh

HumanDFMulticov255=read.table("../data/testQuant/Human_DF_PAS_255.txt")


HumanDFMulticov255_mat=as.matrix(HumanDFMulticov255[,7:12])


#compare this to the earlier one    

HumanDiffMulti=HumanDFMulticov_mat- HumanDFMulticov255_mat

HumanDiffMulti_g=as.data.frame(HumanDiffMulti) %>% gather("Ind", "Count")

ggplot(HumanDiffMulti_g,aes(x=Ind, y=Count, col=Ind)) + geom_boxplot()

Version	Author	Date
02f5c26	brimittleman	2020-03-20

#compare to FC  

HumanDiffw225= HumanCountsFeatureCounts_mat - HumanDFMulticov255_mat
HumanDiffw225_g=as.data.frame(HumanDiffw225) %>% gather("Ind", "Count")
ggplot(HumanDiffw225_g,aes(x=Ind, y=Count, col=Ind)) + geom_boxplot()

Version	Author	Date
02f5c26	brimittleman	2020-03-20

Check examples:

Meta %>% filter(PAS=="chimp171172")

          PAS  disc gene  loc  chr   start     end Chimp Human strandFix
1 chimp171172 Chimp ADI1 utr3 chr2 3497907 3498106 0.155     0         -

HumanDFMulticov %>% filter(V4=="chimp171172")

    V1      V2      V3          V4 V5 V6 V7 V8 V9 V10 V11 V12
1 chr2 3497907 3498106 chimp171172  0  - 24  9 10  15  27 110

HumanDFMulticov255 %>% filter(V4=="chimp171172")

    V1      V2      V3          V4 V5 V6 V7 V8 V9 V10 V11 V12
1 chr2 3497907 3498106 chimp171172  0  -  1  3  0   0   0   0

HumanCountsFeatureCounts %>% filter(PAS=="chimp171172")

   disc         PAS chr2  start2    end2 strand    geneid  Chr   Start
1 Chimp chimp171172 chr2 3497907 3498106      + ADI1_utr3 chr2 3497907
      End Strand Length X18498_N X18499_N X18502_N X18504_N X18510_N
1 3498106      +    200        0        0        0        0        0
  X18523_N
1        0

Ok it seems like maybe multimapping accounts for some of the differece.

Try including the multi map reads in feature counts. I will test the -M flag and the –primary flag to compare. The SAF file has all of the PAS.

sbatch BothFCMM.sh

Grep the results: Chimp:chimp171172:chr2:3497907:3498106:+:ADI1_utr3 chr2 3497907 3498106 + 200 23 6 10 15 27 110

So this is the problem. The IGV.

Inclusive

Test also on the original calls. To see if it can handle this one.

I need to change bed to include both positive and negative. I can do this with python.

python bed2Bedbothstrand.py ../Human/data/inclusivePeaks/human_APApeaks.ALLChrom.bed ../data/testQuant/Human_ALLpeaks.Bothstrand.bed

python bed2Bedbothstrand.py ../Chimp/data/inclusivePeaks/chimp_APApeaks.ALLChrom.bed ../data/testQuant/Chimp_ALLpeaks.Bothstrand.bed

sbatch humanMultiCov_inclusive.sh
sbatch chimpMultiCovInclusive.sh


#look at difference with the -O flag  


sbatch BothFCnewOInclusive.sh

All counts are 0. Fix this.

Address multimaping problem

it looks like the problem is multimapping. I want to get rid of PAS that multi map in one species only. To do this I want to compare the featurecounts with and without the -M flag.


sbatch BothFCMM.sh 


python fixFChead_bothfrac.py ../data/testQuant/ALLPAS_postLift_LocParsed_Human_BothFrac_Mflag ../data/testQuant/ALLPAS_postLift_LocParsed_Human_BothFrac_Mflag_fixed.fc

Only look at the PAS in the double filtered for now. I want to know how many I would lose in both frac.

HumanCountsNoMM=read.table("../Human/data/CleanLiftedPeaks_FC/ALLPAS_postLift_LocParsed_Human_fixed.fc", header = T) %>%  separate(Geneid, into=c("disc", "PAS", "chr2", "start2", "end2", 'strand', 'geneid'), sep=":")%>% filter(PAS %in% Meta$PAS) 

HC_noMM=rowSums(as.matrix(HumanCountsNoMM[,13:24]) )

HumanCountMM=read.table("../data/testQuant/ALLPAS_postLift_LocParsed_Human_BothFrac_Mflag", header = T)%>%  separate(Geneid, into=c("disc", "PAS", "chr2", "start2", "end2", 'strand', 'geneid'), sep=":") %>% filter(PAS %in% Meta$PAS) 


HC_MM=rowSums(as.matrix(HumanCountMM[,13:24]) )

HC_diff=HC_MM-HC_noMM

plot(HC_diff)
abline(120,0,col="red")

Version	Author	Date
02f5c26	brimittleman	2020-03-20

12 libraries. Lets say it needs to have at least 10 in each or 120

sum(HC_diff>120)

[1] 999

ChimpCountsNoMM=read.table("../Chimp/data/CleanLiftedPeaks_FC/ALLPAS_postLift_LocParsed_Chimp_fixed.fc", header = T)  %>%  separate(Geneid, into=c("disc", "PAS", "chr2", "start2", "end2", 'strand', 'geneid'), sep=":") %>% right_join(ChimpMeta,by="PAS")

CC_noMM=rowSums(as.matrix(ChimpCountsNoMM[,13:24]) )

ChimpCountMM=read.table("../data/testQuant/ALLPAS_postLift_LocParsed_Chimp_BothFrac_Mflag", header = T) %>%  separate(Geneid, into=c("disc", "PAS", "chr2", "start2", "end2", 'strand', 'geneid'), sep=":")  %>% right_join(ChimpMeta,by="PAS")


CC_MM=rowSums(as.matrix(ChimpCountMM[,13:24]) )



CC_diff=CC_MM-CC_noMM

plot(CC_diff)
abline(120,0,col="red")

Version	Author	Date
02f5c26	brimittleman	2020-03-20

sum(HC_diff>120)

[1] 999

humanMM=Human=HumanCountsNoMM[which(HC_diff>120),2]
chimpMM=ChimpCountsNoMM[which(CC_diff>120),2]

bothMM=intersect(humanMM,chimpMM)

overlap <- intersect(humanMM, chimpMM) 
H.unique <- setdiff(humanMM,chimpMM) # n 1st NOT 2nd
C.unique <- setdiff(chimpMM,humanMM)
full.list <- unique(c(humanMM,chimpMM))

grid.newpage()
venn.plot <- draw.pairwise.venn(area1 = length(humanMM),
                           area2 = length(chimpMM),
                           cross.area = length(overlap),
                           c("Human", "Chimp"), scaled = TRUE,
                           fill = c("green", "blue"),
                           cex = 1.5,
                           cat.cex = 1.5,
                           cat.pos = c(320, 25),
                           cat.dist = .05)

Version	Author	Date
02f5c26	brimittleman	2020-03-20

total remove if this filter:

length(full.list)

[1] 1528

Test a range of filters:

I will test how many PAS we would lose if we filtered at each value. I will see if there is natural cuttoff.

filterRange=seq(0,500000,by=100)
ChimpDiffN=c()
HumanDiffN=c()

for (i in filterRange){
  ChimpDiffN= c(ChimpDiffN, sum(CC_diff>i))
  HumanDiffN= c(HumanDiffN, sum(HC_diff>i))
}


LostPASdf=as.data.frame(cbind(Filter=filterRange, Chimp=ChimpDiffN, Human=HumanDiffN))
#%>% mutate(All=42318)
LostPASdf_g= LostPASdf %>% gather("Species","PAS_lost", -Filter)

Plot:

ggplot(LostPASdf_g, aes(x=Filter, y=log10(PAS_lost),col=Species)) + geom_point(alpha=.5) + labs(y="log10(PAS lost due to filter)", x="Filter for difference in read count due to multimapping", title="PAS lost due to multimap filter by different filter") + scale_color_brewer(palette = "Dark2")+geom_vline(xintercept = 20000) + annotate("text", label="Difference of 20,000 \ncounts across individuals", x=120000,y=3)

ggplot(LostPASdf_g, aes(x=log10(Filter), y=log10(PAS_lost),col=Species)) + geom_point(alpha=.5) + labs(y="log10(PAS lost due to filter)", x="log10(Filter for difference in read count due to multimapping)", title="PAS lost due to multimap filter by different filter") + scale_color_brewer(palette = "Dark2")

Look at proportion.

LostPASdfPROP=LostPASdf_g%>% mutate(All=42318, propLeft=PAS_lost/All, PropRemain=1-propLeft)

ggplot(LostPASdfPROP, aes(x=Filter, y=PropRemain,col=Species)) + geom_point(alpha=.3)

Zoom in. Look at filter over 1000

LostPASdfPROP_filt=LostPASdfPROP %>% filter(Filter>1000)


ggplot(LostPASdfPROP_filt, aes(x=Filter, y=PropRemain,col=Species)) + geom_point(alpha=.3) + geom_vline(xintercept = 20000) +  annotate("text", label="Difference of 20,000 \ncounts across individuals", x=120000,y=.998) + scale_color_brewer(palette = "Dark2") + labs(x="Filter for difference in reads", y="Proportion of PAS remaining", title="Proportion of PAS remaining by species after multimap filter")

Use this filter to make the boxplot

humanMM2=Human=HumanCountsNoMM[which(HC_diff>20000),2]
chimpMM2=ChimpCountsNoMM[which(CC_diff>20000),2]

bothMM2=intersect(humanMM2,chimpMM2)
overlap2 <- intersect(humanMM2, chimpMM2) 
grid.newpage()
venn.plot <- draw.pairwise.venn(area1 = length(humanMM2),
                           area2 = length(chimpMM2),
                           cross.area = length(overlap2),
                           c("Human", "Chimp"), scaled = TRUE,
                           fill = c("orange", "green"),
                           cex = 1.5,
                           cat.cex = 1.5,
                           cat.pos = c(320, 25),
                           cat.dist = .05)

expect to loose chimp171173 in human

HumanCountsNoMM[HumanCountsNoMM$PAS=="chimp171173",]

       disc         PAS chr2  start2    end2 strand    geneid  Chr   Start
22206 Chimp chimp171173 chr2 3498436 3498639      + ADI1_utr3 chr2 3498436
          End Strand Length X18498_N X18498_T X18499_N X18499_T X18502_N
22206 3498639      +    204        0        0        0        0        0
      X18502_T X18504_N X18504_T X18510_N X18510_T X18523_N X18523_T
22206        0        0        0        0        1        0        0

HC_diff[22206]

[1] 3808

Should it be the proportion of reads lost?

I need the sum for the reads with and without multimapping at each PAS. Proportion of reads in the mulimap set

HumanPASMultiMap=as.data.frame(cbind(PAS=HumanCountMM$PAS,WithMultiMap=HC_MM, NoMultiMap=HC_noMM)) 
HumanPASMultiMap$WithMultiMap=as.numeric(as.character(HumanPASMultiMap$WithMultiMap))
HumanPASMultiMap$NoMultiMap=as.numeric(as.character(HumanPASMultiMap$NoMultiMap))
HumanPASMultiMapProp= HumanPASMultiMap%>% mutate(Difference=WithMultiMap-NoMultiMap, PercentLost=Difference/WithMultiMap)

check chimp171173

HumanPASMultiMapProp %>% filter(PAS=="chimp171173")

          PAS WithMultiMap NoMultiMap Difference PercentLost
1 chimp171173         3809          1       3808   0.9997375

Plot
Color top 10%

x=quantile(HumanPASMultiMapProp$PercentLost, seq(0,1, by=.05),na.rm = T)[20]

HumanPASMultiMapPropcol=HumanPASMultiMapProp %>% mutate(color=ifelse(PercentLost>=x, "red", "black")) %>% arrange(PercentLost)

plot(HumanPASMultiMapPropcol$PercentLost,col=HumanPASMultiMapPropcol$color,ylab = "Percent of reads multimapping",main="Percent of reads lost to multimap filter in each human PAS",xlab="42,318 PAS Sorted")
legend("topleft", legend=c("Bottom 95%", "Top 5%"),col=c("black", "red"), pch=16,bty = 'n')

Look at this in chimp

ChimpPASMultiMap=as.data.frame(cbind(PAS=ChimpCountMM$PAS,WithMultiMap=CC_MM, NoMultiMap=CC_noMM)) 
ChimpPASMultiMap$WithMultiMap=as.numeric(as.character(ChimpPASMultiMap$WithMultiMap))
ChimpPASMultiMap$NoMultiMap=as.numeric(as.character(ChimpPASMultiMap$NoMultiMap))
ChimpPASMultiMapProp= ChimpPASMultiMap%>% mutate(Difference=WithMultiMap-NoMultiMap, PercentLost=Difference/WithMultiMap)

Plot
Color top 5%

y=quantile(ChimpPASMultiMapProp$PercentLost, seq(0,1, by=.05),na.rm = T)[20]

ChimpPASMultiMapPropcol=ChimpPASMultiMapProp %>% mutate(color=ifelse(PercentLost>=y, "red", "black")) %>% arrange(PercentLost)

plot(ChimpPASMultiMapPropcol$PercentLost,col=ChimpPASMultiMapPropcol$color,ylab = "Percent of reads multimapping",main="Percent of reads lost to multimap filter in each chimp PAS",xlab="42,318 PAS Sorted")
legend("topleft", legend=c("Bottom 95%", "Top 5%"),col=c("black", "red"), pch=16,bty = 'n')

Are these the same PAS :

Look at example.

ChimpPASMultiMapProp %>% filter(PAS=="chimp171173")

          PAS WithMultiMap NoMultiMap Difference PercentLost
1 chimp171173         2219       2216          3  0.00135196

So this shows that this PAS would not be lost in chimp

ChimpTop5=ChimpPASMultiMapPropcol %>% filter(color=="red") %>% select(PAS)
HumanTop5=HumanPASMultiMapPropcol %>% filter(color=="red") %>% select(PAS)

bothLostProp=intersect(HumanTop5$PAS,ChimpTop5$PAS)
OverlapProp <-intersect(HumanTop5$PAS, ChimpTop5$PAS) 
grid.newpage()
venn.plot <- draw.pairwise.venn(area1 = length(HumanTop5$PAS),
                           area2 = length(ChimpTop5$PAS),
                           cross.area = length(OverlapProp),
                           c("Human", "Chimp"), scaled = TRUE,
                           fill = c("darkorange", "darkgreen"),
                           cex = 1.5,
                           cat.cex = 1.5,
                           cat.pos = c(320, 25),
                           cat.dist = .05)

This shows that multimapping is not equivalent between.

Color top 2% (get up to 13% difference )

y=quantile(ChimpPASMultiMapProp$PercentLost, seq(0,1, by=.01),na.rm = T)[99]

ChimpPASMultiMapPropcol2=ChimpPASMultiMapProp %>% mutate(color=ifelse(PercentLost>=y, "red", "black")) %>% arrange(PercentLost)

x=quantile(HumanPASMultiMapProp$PercentLost, seq(0,1, by=.01),na.rm = T)[99]

HumanPASMultiMapPropcol2=HumanPASMultiMapProp %>% mutate(color=ifelse(PercentLost>=y, "red", "black")) %>% arrange(PercentLost)

plot(HumanPASMultiMapPropcol2$PercentLost,col=HumanPASMultiMapPropcol2$color,ylab = "Percent of reads multimapping",main="Percent of reads lost to multimap filter in each human PAS",xlab="42,318 PAS Sorted")
legend("topleft", legend=c("Bottom 98%", "Top 2%"),col=c("black", "red"), pch=16,bty = 'n')

plot(ChimpPASMultiMapPropcol2$PercentLost,col=ChimpPASMultiMapPropcol2$color,ylab = "Percent of reads multimapping",main="Percent of reads lost to multimap filter in each chimp PAS",xlab="42,318 PAS Sorted")
legend("topleft", legend=c("Bottom 98%", "Top 2%"),col=c("black", "red"), pch=16,bty = 'n')

ChimpTop2=ChimpPASMultiMapPropcol2 %>% filter(color=="red") %>% select(PAS)
HumanTop2=HumanPASMultiMapPropcol2 %>% filter(color=="red") %>% select(PAS)

bothLostProp=intersect(HumanTop2$PAS,ChimpTop2$PAS)
OverlapProp <-intersect(HumanTop2$PAS, ChimpTop2$PAS) 
grid.newpage()
venn.plot <- draw.pairwise.venn(area1 = length(HumanTop2$PAS),
                           area2 = length(ChimpTop2$PAS),
                           cross.area = length(OverlapProp),
                           c("Human", "Chimp"), scaled = TRUE,
                           fill = c("darkorange", "darkgreen"),
                           cex = 1.5,
                           cat.cex = 1.5,
                           cat.pos = c(320, 25),
                           cat.dist = .05)

In this set there are more on the chimp side.

Will need to choose a cutoff.

sessionInfo()

R version 3.5.1 (2018-07-02)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Scientific Linux 7.4 (Nitrogen)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] grid      stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
 [1] Matrix_1.2-15       VennDiagram_1.6.20  futile.logger_1.4.3
 [4] ggpubr_0.2          magrittr_1.5        forcats_0.3.0      
 [7] stringr_1.3.1       dplyr_0.8.0.1       purrr_0.3.2        
[10] readr_1.3.1         tidyr_0.8.3         tibble_2.1.1       
[13] ggplot2_3.1.1       tidyverse_1.2.1    

loaded via a namespace (and not attached):
 [1] tidyselect_0.2.5     haven_1.1.2          lattice_0.20-38     
 [4] colorspace_1.3-2     generics_0.0.2       htmltools_0.3.6     
 [7] yaml_2.2.0           rlang_0.4.0          later_0.7.5         
[10] pillar_1.3.1         glue_1.3.0           withr_2.1.2         
[13] RColorBrewer_1.1-2   lambda.r_1.2.3       modelr_0.1.2        
[16] readxl_1.1.0         plyr_1.8.4           munsell_0.5.0       
[19] gtable_0.2.0         workflowr_1.6.0      cellranger_1.1.0    
[22] rvest_0.3.2          evaluate_0.12        labeling_0.3        
[25] knitr_1.20           httpuv_1.4.5         broom_0.5.1         
[28] Rcpp_1.0.2           formatR_1.5          promises_1.0.1      
[31] scales_1.0.0         backports_1.1.2      jsonlite_1.6        
[34] fs_1.3.1             hms_0.4.2            digest_0.6.18       
[37] stringi_1.2.4        rprojroot_1.3-2      cli_1.1.0           
[40] tools_3.5.1          lazyeval_0.2.1       futile.options_1.0.1
[43] crayon_1.3.4         whisker_0.3-2        pkgconfig_2.0.2     
[46] xml2_1.2.0           lubridate_1.7.4      assertthat_0.2.0    
[49] rmarkdown_1.10       httr_1.3.1           rstudioapi_0.10     
[52] R6_2.3.0             nlme_3.1-137         git2r_0.26.1        
[55] compiler_3.5.1

Try different quantification methods

Briana Mittleman

3/19/2020

Inclusive

Address multimaping problem