Location of Nuclear Intronic PAS
Briana Mittleman
5/15/2019
Last updated: 2019-05-23
Checks: 6 0
Knit directory: apaQTL/analysis/
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| File | Version | Author | Date | Message |
|---|---|---|---|---|
| Rmd | 2632f11 | brimittleman | 2019-05-23 | add tot enriched |
| html | b454fc6 | brimittleman | 2019-05-23 | Build site. |
| Rmd | fc5727e | brimittleman | 2019-05-23 | add boxplots by pau |
| html | ab26926 | brimittleman | 2019-05-22 | Build site. |
| Rmd | 6bfa078 | brimittleman | 2019-05-22 | by delta pau |
| html | ace11cc | brimittleman | 2019-05-22 | Build site. |
| html | 57d8a8c | brimittleman | 2019-05-22 | Build site. |
| Rmd | a259a17 | brimittleman | 2019-05-22 | fix bug with utrs |
| html | 365e817 | brimittleman | 2019-05-21 | Build site. |
| html | d859f02 | brimittleman | 2019-05-21 | Build site. |
| Rmd | 82fdc65 | brimittleman | 2019-05-21 | add by length |
| html | 801ca1b | brimittleman | 2019-05-20 | Build site. |
| Rmd | a455701 | brimittleman | 2019-05-20 | analysis plot |
| html | d89772d | brimittleman | 2019-05-15 | Build site. |
| Rmd | ee92964 | brimittleman | 2019-05-15 | start ideas for inton analysis |
I am interested in understanding where in the introns the nuclear peaks are. Are they closer to the three prime or five prime edge of the intron. This may help us understand if NMD is contributing to the loss of transcripts between the nuclear and total fraction.
I need to create an annotation with introns that do not overlap. For this I will use line up all of the exons for a gene then take the open spaces as introns.
library(tidyverse)── Attaching packages ───────────────────────────────────────────────── tidyverse 1.2.1 ──✔ ggplot2 3.1.1 ✔ purrr 0.3.2
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✖ dplyr::filter() masks stats::filter()
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Run ?workflowr for help getting startedlibrary(cowplot)
Attaching package: 'cowplot'The following object is masked from 'package:ggplot2':
ggsavenucIntronicPeaks=read.table("../data/peaks_5perc/APApeak_Peaks_GeneLocAnno.Nuclear.5perc.fc", stringsAsFactors = F, header = F,col.names = c("chr", "start", "end", "gene", "loc", "strand", "peak", "avgUsage")) %>% filter(loc=="intron")
nucIntronicPeaksBed=nucIntronicPeaks %>% mutate(ID=paste(peak,gene,loc, sep=":")) %>% dplyr::select(chr, start, end, ID, avgUsage, strand)
write.table(nucIntronicPeaksBed, "../data/intron_analysis/NuclearIntronicPeaks.bed", col.names = F, row.names = F, quote = F,sep="\t")I will need to assign each of these to an intron in the new annotation.
The genome annotation file, Transcript2GeneName.dms has the information i need. I will need to parse this file. I need all exons for a gene (longest transcript) The file has the exon starts and ends for each transcript.
I will remove the exon locations for full transcripts using bedtools subtract.
Create transcript file.I will select all of the transcripts in the dms file and merge by gene name. Then I can subtract the exons
python transcriptdm2bed.pySort the output, group by transcript and fix order of columns.
sort -k1,1 -k2,2n /project2/gilad/briana/genome_anotation_data/RefSeq_annotations/AllTranscriptsbyName.bed > /project2/gilad/briana/genome_anotation_data/RefSeq_annotations/AllTranscriptsbyName.sort.bed
sbatch grouptranscripts.py
python fixgroupedtranscript.pyI want to subract any exon or UTR seqeunce. I have an annotation bed file I will use:
exonandUTRs=read.table("../../genome_anotation_data/RefSeq_annotations/ncbiRefSeq_FormatedallAnnotation.sort.bed", col.names = c("CHR", "start", "end", "ID", "score", "strand"),stringsAsFactors = F)%>% separate(ID, into=c("loc", "gene"),sep=":") %>% filter(loc!="intron") %>% dplyr::select(-loc) %>% mutate(CHR=paste("chr", CHR, sep=""))
write.table(exonandUTRs, file="../data/intron_analysis/ExonandUTRloc.bed", quote=F, col.names = F, row.names = F, sep="\t")sort -k1,1 -k2,2n ../data/intron_analysis/ExonandUTRloc.bed > ../data/intron_analysis/ExonandUTRloc.sort.bedsbatch subtractExons.shsort:
sort -k1,1 -k2,2n /project2/gilad/briana/apaQTL/data/intron_analysis/transcriptsMinusExons.bed > /project2/gilad/briana/apaQTL/data/intron_analysis/transcriptsMinusExons.sort.bedNext I will map the intronic peaks on these positions.
sbatch assignNucIntonpeak2intronlocs.shPlot percentage of intron where PAS is.
pas2intron=read.table("../data/intron_analysis/IntronPeaksontoIntrons.bed",col.names = c("intronCHR", "intronStart", "intronEnd", "gene", "score", "strand", "peakCHR", "peakStart", "peakEnd", "PeakID", "meanUsage", "peakStrand")) %>% mutate(PASloc=ifelse(strand=="+", peakEnd, peakStart)) %>% dplyr::select(intronStart, intronEnd, gene, strand, PeakID, PASloc ,meanUsage) %>% mutate(intronLength=intronEnd-intronStart , distance2PAS= ifelse(strand=="+", PASloc-intronStart, intronEnd-PASloc), propIntron=distance2PAS/intronLength)nuclearplot=ggplot(pas2intron, aes(x=propIntron)) + geom_histogram(bins=50, aes(y=..count../33345)) + labs(title="PAS position within intron \n for Nuclear PAS", y="Proportion of Intronic PAS", x="Proportion of Intronic Length")Facet by usage 0-25, 25-50, 50-75, 75-1
pas2intron_usage=pas2intron %>% mutate(UsageCat=ifelse(meanUsage<=.5, "low", "high"))
ggplot(pas2intron_usage, aes(x=propIntron, fill=UsageCat)) + geom_histogram(bins=50, aes(y=..count../33345)) + labs(title="PAS position within intron", y="Proportion of Intronic PAS", x="Proportion of Intronic Length") + facet_grid(~UsageCat)
Look at different intron lengths:
First i want to look at the distribution of intorn lengths:
ggplot(pas2intron_usage, aes(x=log10(intronLength))) + geom_density()
I will look at above and below the mean intron length:
meanIntronlength=mean(pas2intron_usage$intronLength)
pas2intron_length=pas2intron %>% mutate(LengthCat=ifelse(intronLength<=meanIntronlength, "bottom", "top"))
ggplot(pas2intron_length, aes(x=propIntron, fill=LengthCat)) + geom_histogram(bins=50, aes(y=..count../33345)) + labs(title="PAS position within intron", y="Proportion of Intronic PAS", x="Proportion of Intronic Length") + facet_grid(~LengthCat)
| Version | Author | Date |
|---|---|---|
| 57d8a8c | brimittleman | 2019-05-22 |
ggplot(pas2intron_length, aes(x=distance2PAS, fill=LengthCat)) + geom_histogram(bins=50, aes(y=..count../33345)) + labs(title="PAS position within intron", y="Proportion of Intronic PAS", x="Proportion of Intronic Length") + facet_grid(~LengthCat)
| Version | Author | Date |
|---|---|---|
| 57d8a8c | brimittleman | 2019-05-22 |
Look at quartiles:
summary(pas2intron_usage$intronLength) Min. 1st Qu. Median Mean 3rd Qu. Max.
106 3929 9220 22248 24094 1102540 pas2intron_length2=pas2intron %>% mutate(LengthCat=ifelse(intronLength<=3929, "first", ifelse(intronLength>3929 &intronLength<=9220, "second", ifelse(intronLength>9220 &intronLength<=24094, "third", "fourth"))))
pas2intron_length2$LengthCat <- factor(pas2intron_length2$LengthCat, levels=c("first", "second", "third", "fourth"))
ggplot(pas2intron_length2, aes(x=propIntron, fill=LengthCat)) + geom_histogram(bins=50, aes(y=..count../33345)) + labs(title="PAS position within intron \n Nuclear intronic PAS", y="Proportion of Intronic PAS", x="Proportion of Intronic Length") + facet_grid(~LengthCat)+theme(axis.text.x = element_text(angle = 90, hjust = 1))
ggplot(pas2intron_length2, aes(x=log(distance2PAS)), by=LengthCat, col=LengthCat) + stat_ecdf(aes(col=LengthCat)) 
Look at distribution in total fraction:
totIntronicPeaks=read.table("../data/peaks_5perc/APApeak_Peaks_GeneLocAnno.Total.5perc.fc", stringsAsFactors = F, header = F,col.names = c("chr", "start", "end", "gene", "loc", "strand", "peak", "avgUsage")) %>% filter(loc=="intron")
totIntronicPeaksBed=totIntronicPeaks %>% mutate(ID=paste(peak,gene,loc, sep=":")) %>% dplyr::select(chr, start, end, ID, avgUsage, strand)
write.table(totIntronicPeaksBed, "../data/intron_analysis/TotalIntronicPeaks.bed", col.names = F, row.names = F, quote = F,sep="\t")map these to the intron file
sbatch assignTotIntronpeak2intronlocs.shpas2intronTot=read.table("../data/intron_analysis/TotalIntronPeaksontoIntrons.bed",col.names = c("intronCHR", "intronStart", "intronEnd", "gene", "score", "strand", "peakCHR", "peakStart", "peakEnd", "PeakID", "meanUsage", "peakStrand")) %>% mutate(PASloc=ifelse(strand=="+", peakEnd, peakStart)) %>% dplyr::select(intronStart, intronEnd, gene, strand, PeakID, PASloc ,meanUsage) %>% mutate(intronLength=intronEnd-intronStart , distance2PAS= ifelse(strand=="+", PASloc-intronStart, intronEnd-PASloc), propIntron=distance2PAS/intronLength)
nrow(pas2intronTot)[1] 31954totalplot=ggplot(pas2intronTot, aes(x=propIntron)) + geom_histogram(bins=50, aes(y=..count../31954)) + labs(title="PAS position within intron \nfor total PAS", y="Proportion of Intronic PAS", x="Proportion of Intronic Length")plot_grid(totalplot, nuclearplot)
| Version | Author | Date |
|---|---|---|
| 57d8a8c | brimittleman | 2019-05-22 |
summary(pas2intronTot$intronLength) Min. 1st Qu. Median Mean 3rd Qu. Max.
106 3785 8872 21032 22928 1102540 pas2intron_totlength2=pas2intronTot %>% mutate(LengthCat=ifelse(intronLength<=3785, "first", ifelse(intronLength>3785 &intronLength<=8872, "second", ifelse(intronLength>8872 &intronLength<=22928, "third", "fourth"))))
pas2intron_totlength2$LengthCat <- factor(pas2intron_totlength2$LengthCat, levels=c("first", "second", "third", "fourth"))
ggplot(pas2intron_totlength2, aes(x=propIntron, fill=LengthCat)) + geom_histogram(bins=50, aes(y=..count../31954)) + labs(title="PAS position within intron \n Total intronic PAS", y="Proportion of Intronic PAS", x="Proportion of Intronic Length") + facet_grid(~LengthCat) + theme(axis.text.x = element_text(angle = 90, hjust = 1))
Look by differences in \(\Delta\) PAU.
effectsize=read.table("../data/DiffIso/TN_diff_isoform_AllChrom_effect_sizes.txt", stringsAsFactors = F, col.names=c('PAS', 'logef' ,'Nuclear', 'Total','deltaPAU')) %>% filter(PAS != "intron") %>% dplyr::select(PAS, deltaPAU)pas2intronNuc=read.table("../data/intron_analysis/IntronPeaksontoIntrons.bed",col.names = c("intronCHR", "intronStart", "intronEnd", "gene", "score", "strand", "peakCHR", "peakStart", "peakEnd", "PeakID", "meanUsage", "peakStrand")) %>% mutate(PAS=paste(peakCHR, peakStart, peakEnd, gene, sep=":")) %>% inner_join(effectsize, by="PAS") %>% mutate(PASloc=ifelse(strand=="+", peakEnd, peakStart)) %>% dplyr::select(intronStart, intronEnd, gene, strand, PeakID, PASloc ,meanUsage, deltaPAU) %>% mutate(intronLength=intronEnd-intronStart , distance2PAS= ifelse(strand=="+", PASloc-intronStart, intronEnd-PASloc), propIntron=distance2PAS/intronLength)
pas2intronNuc$deltaPAU= as.numeric(pas2intronNuc$deltaPAU)Plot relationship between delta PAU and distance to proporiton of intron
ggplot(pas2intronNuc, aes(x=propIntron, y=deltaPAU)) + geom_point() + geom_density2d(col="red")
| Version | Author | Date |
|---|---|---|
| ab26926 | brimittleman | 2019-05-22 |
pas2intronNuc_cat=pas2intronNuc %>% mutate(Fraction=ifelse(deltaPAU <= -0.2, "NuclearSpecific", ifelse(deltaPAU>-.2 &deltaPAU < .2, "NotDiff", "TotalEnriched"))) %>% mutate(LengthCat=ifelse(intronLength<=3929, "first", ifelse(intronLength>3929 &intronLength<=9220, "second", ifelse(intronLength>9220 &intronLength<=24094, "third", "fourth"))))
pas2intronNuc_cat$LengthCat <- factor(pas2intronNuc_cat$LengthCat, levels=c("first", "second", "third", "fourth"))Plot this:
ggplot(pas2intronNuc_cat, aes(y=propIntron, x=Fraction, fill=Fraction)) + geom_boxplot(alpha=.5) + facet_grid(~LengthCat) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) 
ggplot(pas2intronNuc_cat, aes(x=propIntron, by=Fraction, fill=Fraction)) + geom_density(alpha=.5) + facet_grid(~LengthCat) + theme(axis.text.x = element_text(angle = 90, hjust = 1))
| Version | Author | Date |
|---|---|---|
| b454fc6 | brimittleman | 2019-05-23 |
ggplot(pas2intronNuc_cat, aes(y=propIntron, x=Fraction, fill=Fraction)) + geom_boxplot(alpha=.5) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) 
pas2intronTotal=read.table("../data/intron_analysis/TotalIntronPeaksontoIntrons.bed",col.names = c("intronCHR", "intronStart", "intronEnd", "gene", "score", "strand", "peakCHR", "peakStart", "peakEnd", "PeakID", "meanUsage", "peakStrand")) %>% mutate(PAS=paste(peakCHR, peakStart, peakEnd, gene, sep=":")) %>% inner_join(effectsize, by="PAS") %>% mutate(PASloc=ifelse(strand=="+", peakEnd, peakStart)) %>% dplyr::select(intronStart, intronEnd, gene, strand, PeakID, PASloc ,meanUsage, deltaPAU) %>% mutate(intronLength=intronEnd-intronStart , distance2PAS= ifelse(strand=="+", PASloc-intronStart, intronEnd-PASloc), propIntron=distance2PAS/intronLength)
pas2intronTotal$deltaPAU= as.numeric(pas2intronTotal$deltaPAU)
pas2intronTotal_cat=pas2intronTotal %>% mutate(Fraction=ifelse(deltaPAU <= -0.2, "NuclearSpecific", ifelse(deltaPAU>-.2 &deltaPAU < .2, "NotDiff", "TotalEnriched"))) %>% mutate(LengthCat=ifelse(intronLength<=3785, "first", ifelse(intronLength>3785 &intronLength<=8872, "second", ifelse(intronLength>8872 &intronLength<=22928, "third", "fourth"))))
pas2intronTotal_cat$LengthCat <- factor(pas2intronTotal_cat$LengthCat, levels=c("first", "second", "third", "fourth"))
ggplot(pas2intronTotal_cat, aes(y=propIntron, x=Fraction, fill=Fraction)) + geom_boxplot(alpha=.5) + facet_grid(~LengthCat) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) 
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] stats graphics grDevices utils datasets methods base
other attached packages:
[1] cowplot_0.9.4 workflowr_1.3.0 forcats_0.3.0 stringr_1.3.1
[5] dplyr_0.8.0.1 purrr_0.3.2 readr_1.3.1 tidyr_0.8.3
[9] tibble_2.1.1 ggplot2_3.1.1 tidyverse_1.2.1
loaded via a namespace (and not attached):
[1] Rcpp_1.0.0 cellranger_1.1.0 pillar_1.3.1 compiler_3.5.1
[5] git2r_0.23.0 plyr_1.8.4 tools_3.5.1 digest_0.6.18
[9] lubridate_1.7.4 jsonlite_1.6 evaluate_0.12 nlme_3.1-137
[13] gtable_0.2.0 lattice_0.20-38 pkgconfig_2.0.2 rlang_0.3.1
[17] cli_1.0.1 rstudioapi_0.10 yaml_2.2.0 haven_1.1.2
[21] withr_2.1.2 xml2_1.2.0 httr_1.3.1 knitr_1.20
[25] hms_0.4.2 generics_0.0.2 fs_1.2.6 rprojroot_1.3-2
[29] grid_3.5.1 tidyselect_0.2.5 glue_1.3.0 R6_2.3.0
[33] readxl_1.1.0 rmarkdown_1.10 reshape2_1.4.3 modelr_0.1.2
[37] magrittr_1.5 whisker_0.3-2 MASS_7.3-51.1 backports_1.1.2
[41] scales_1.0.0 htmltools_0.3.6 rvest_0.3.2 assertthat_0.2.0
[45] colorspace_1.3-2 labeling_0.3 stringi_1.2.4 lazyeval_0.2.1
[49] munsell_0.5.0 broom_0.5.1 crayon_1.3.4