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6.2.APA (Alternative Polyadenylation) Detection

1) Workflow

2) Background

Alternative polyadenylation (APA) leading to the production of two mRNA isoforms with different 3ʹ untranslated regions (3ʹ UTRs)The dynamic usage of the 3’untranslated region (3’UTR) resulting from alternative polyadenylation (APA) is emerging as a pervasive mechanism for regulating mRNA diversity, stability and translation.

3) Running steps (DaPars)

启动 6.2 APA, 6.3 Ribo-seq, 6.4 Structure-seq的 Docker，然后进入工作目录

cd /home/test/rna_regulation/apa

3a) Generate region annotation

DaPars will use the extracted distal polyadenylation sites to infer the proximal polyadenylation sites based on the alignment wiggle files of two samples. The output in this step will be used by the next step.

starting analysis

DaPars_Extract_Anno.py -b hg19_refseq_whole_gene.bed -s hg19_4_19_2012_Refseq_id_from_UCSC.txt -o hg19_refseq_extracted_3UTR.bed

input

hg19_refseq_whole_gene.bed (bed12 format)

   chr1    66999824    67210768    NM_032291    0    +    67000041    67208778    25    227,64,25,72,57,55,176,12,12,25,52,86,93,75,501,128,127,60,112,156,133,203,65,165,2013,    0,91705,98928,101802,105635,108668,109402,126371,133388,136853,137802,139139,142862,145536,147727,155006,156048,161292,185152,195122,199606,205193,206516,207130,208931,
   chr1    33546713    33585995    NM_052998    0    +    33547850    33585783    12    182,121,212,177,174,173,135,166,163,113,215,351,    0,275,488,1065,2841,10937,12169,13435,15594,16954,36789,38931,
   chr1    16767166    16786584    NM_001145278    0    +    16767256    16785385    104,101,105,82,109,178,76,1248,    0,2960,7198,7388,8421,11166,15146,18170,

hg19_4_19_2012_Refseq_id_from_UCSC.txt

   #name    name2
   NM_032291    SGIP1
   NM_052998    ADC

output

hg19_refseq_extracted_3UTR.bed

   chr14    50792327    50792946    NM_001003805|ATP5S|chr14|+    0    +
   chr9    95473645    95477745    NM_001003800|BICD2|chr9|-    0    -
   chr11    92623657    92629635    NM_001008781|FAT3|chr11|+    0    +

3b) Main function to get final result

starting analysis

DaPars_main.py configure_file

Run this function to get the final result. The configure file is the only parameter for DaPars_main.py, which stores all the parameters.

input

configure_file

The format of the configure file is:

#The following file is the result of step 1.

Annotated_3UTR=hg19_refseq_extracted_3UTR.bed

#A comma-separated list of BedGraph files of samples from condition 1

Group1_Tophat_aligned_Wig=Condition_A_chrX.wig
#Group1_Tophat_aligned_Wig=Condition_A_chrX_r1.wig,Condition_A_chrX_r2.wig if multiple files in one group

#A comma-separated list of BedGraph files of samples from condition 2

Group2_Tophat_aligned_Wig=Condition_B_chrX.wig

Output_directory=DaPars_Test_data/

Output_result_file=DaPars_Test_data

#At least how many samples passing the coverage threshold in two conditions
Num_least_in_group1=1

Num_least_in_group2=1

Coverage_cutoff=30

#Cutoff for FDR of P-values from Fisher exact test.

FDR_cutoff=0.05


PDUI_cutoff=0.5

Fold_change_cutoff=0.59

output

3c) Filter diff-APA events

FDR_cutoff, PDUI_cutoff, Fold_change_cutoff → Pass filer (Y nor N)

4) Homework

运行示例文件，理解输出文件“DaPars_Test_data_All_Prediction_Results.txt”中每一列的含义。 (1)解释PDUI的含义； (2)写脚本过滤adjusted.P_val<=0.05,PDUI_Group_diff>=0.5, PDUI_fold_change>=0.59的作为diff-APA events，和Pass_filter为“Y“筛选出来的diff-APA events做比较。