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Prediction of APA sites and APA dynamics using different tools.

Example files of input data

  • Brain.fq (MAQC Brain exp 2 phi X) & Brian_control.fq (MAQC UHR exp 2 auto) -- RNA-seq datasets from two conditions
  • hg19.fa -- reference genome sequence
  • hg19.gff/hg19_refFlat.txt -- reference genome annotation

Running different tools for APA prediction

  • Generate bam file and bedgraph file

    • Use Trimmomatic to trim and filter out low quality raw reads.
      java -jar trimmomatic-0.35.jar SE -phred33 Brain.fq  Brain.paired.fastq Brain.unpaired.fastq ILLUMINACLIP:TruSeq3-SE-2.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
    • Use HISAT2 or tophat to align the Brain/Brain_control reads to the reference genome.
      hisat2 -x reference_genome_index Brain.paired.fastq -S Brain.sam

      samtools view -bS Brain.sam > Brain.bam  

  samtools sort Brain.bam -o Brain.sorted.bam  

  samtools index Brain.sorted.bam  

  genomeCoverageBed -bg -ibam Brain.sort.bam -g reference.genome.size.txt -split > Brain.bedgraph

  • MISO

    • Index the annotation using index_gff
      index_gff --index hg19.gff  indexed/

    where refGene.gff3 is a GFF file containing descriptions of isoforms/alternative splicing events to be quantitated (e.g. skipped exons)

    • Run MISO
    miso --run indexed/  Brain.bam --output-dir output1/  --read-len 50 –use-cluster
miso --run indexed/  Brain_control.bam --output-dir output2/  --read-len 50 –use-cluster

    The --read-len option is necessary and specifies the length of the reads in the data
    To compute expression levels using paired-end reads, use the --paired-end option

    • Summarize MISO inferences using summarize_miso --summarize-samples
    summarize_miso --summarize-samples output1/ summary_output1/
summarize_miso --summarize-samples output2/ summary_output2/
    • Make pairwise comparisons between samples to detect differentially expressed isoforms/events with compare_miso --compare-samples
    compare_miso --compare-samples output1/ output2/ comparison_output/

  • roar

    library(roar)
gtf <- system.file("examples", "apa.gtf", package="roar")
bamTreatment <- c("Brain.bam")
bamControl <- c("Brain_control.bam")
rds <- RoarDatasetFromFiles(bamTreatment, bamControl, gtf)
rds <- countPrePost(rds, FALSE)
rds <- computeRoars(rds)
rds <- computePvals(rds)
results <- totalResults(rds)
results_filtered <- pvalueFilter(rds, fpkmCutoff=-Inf,  pvalCutoff=0.05

  • QAPA

    • Extract 3′ UTRs from annotation
    qapa build --db ensembl_identifiers.txt -g gencode.polyA_sites.bed -p clusters.mm10.bed gencode.basic.txt > output_utrs.bed

    If using a custom BED file, replace the -g and -p options with -o:

    qapa build --db ensembl_identifiers.txt -o custom_sites.bed gencode.basic.txt > output_utrs.bed
    • Extract sequences from the BED file prepared by build (a reference genome in FASTA format is required)
    qapa fasta -f hg19.fa output_utrs.bed output_sequences.fa

    hg19.fa must be uncompressed

    • Expression quantification of 3’UTR isoforms must be carried out first using the FASTA file prepared by fasta as the index
      To index the sequences using Salmon
    salmon index -t output_sequences.fa -i utr_library

    next

    qapa quant --db ensembl_identifiers.txt project/sample*/quant.sf > pau_results.txt

  • PAQR

    • Configure the input parameters
      The PAQR subdirectory contains a file called "config.yaml". This files contains all information about used parameter values, data locations, file names and so on. During a run, all steps of the pipeline will retrieve their parameter values from this file.
    max_cores=4 # maximum number of threads that will run in parallel
snakemake -s part_one.Snakefile -p --cores ${max_cores} &> log_output.log

    max_cores=8 # maximum number of threads that will run in parallel
snakemake -s part_two.Snakefile -p --cores ${max_cores} &>> log_output.log
    • The single steps/scripts of the pipeline
    python calculate-TIN-values.py \
       -i data/bam_files/Brain.bam \
       -r ${transcripts} \
       -c ${min_raw_reads} \
       --names KD_rep1 \
       -n ${sample_size} \
       > data/Brain.tsv

    python merge-TIN-tables.py \
       --verbose \
       --input data/Brain.tsv data/Brain_control.tsv \
       > data/bias.transcript_wide.TIN.tsv

    python merge-TIN-tables.py \
       --verbose \
       --input data/Brain.tsv data/Brain_control.tsv \
       > data/bias.transcript_wide.TIN.tsv

    bias.transcript_wide.TIN.tsv > bias.TIN.median_per_sample.tsv in part_one.Snakefile

    Rscript boxplots-TIN-distributions.R \
        --file HNRNPC_KD/bias.TIN.median_per_sample.tsv\
        --pdf HNRNPC_KD/bias.transcript_wide.TIN.boxplots.pdf
    • Run KAPAC
    Rscript --vanilla KAPAC.R --help

  • Cufflinks

    cufflinks -p 8 -o brain --overlap-radius 75 brain.sorted.bam

    cuffdiff -o ./cuffdiff_out/ -p 2 -FDR 0.1 -L c1,c2 -max-bundle-frags 20000000 hg19.gtf Brain.sorted.bam Brain_control.sorted.bam

  • ExUTR

    • Reading Open Frame (ORF) prediction
    perl 3UTR_orf.pl -i transcripts.fasta  -d /home/user/swissprot/swissprot -a 8 -o Test -l un
    • 3'UTR sequence retrieval
    perl 3UTR_ext.pl -i1 transcripts.fa -i2 orfs.fasta -d /home/user/3UTR_database/3UTR.mam.fasta -a 8 -o 3UTR.fasta -x 2500 -m 20

  • Scripture

    • Make paired end alignment files using Scripture
      Remove the headers from the TopHat alignment file (headers begin with "@") and sort each by read name
    sed '1,2d' Brian.sam | sort > Brian.sorted.sam
sed '1,2d' Brian_control.sam | sort > Brian_control.sorted.sam

    java -Xmx4000m -jar scripture.jar -task makePairedFile -pair1 Brain.sorted.sam -pair2 Brian_control.sam -out Brain.paired.sam –sorted
    • Run Scripture
      Combine TopHat alignment file and paired end alignments file, then sort and index
    cat Brain.sorted.sam Brian_control.sorted.sam > all_Brain.sam

    cat Brain.paired.sam Brain2.paired.sam > all_Brain.paired.sam

    java –jar scripture.jar –alignment all_Brain.sorted.sam –out Brain_Test –sizeFile hg19.sizes –chr chr19 –chrSequence chr19.fa -pairedEnd all_Brain.paired.sorted.sam

  • KLEAT

    • Use the included script (TA.sh) to generate the input necessary for KLEAT
     TA.sh -a Brain1.fq.gz -b Brain2.fq.gz -n Brain -k "32 52 72" -o Brain/assembly -t 6 -m 15G
    • Run KLEAT
     python KLEAT.py  Brain/assembly/Brain.bam Brain/assembly/merged/Brain-merged.fa hg19.fa ensembl.fixed.sorted.gz Brain/assembly/r2c_sorted.bam /KLEAT/Brain -k KLEAT_Brain "KLEAT cleavage sites" -ss

  • ContextMap2

    java -jar ContextMap_v2.1.0.jar mapper -read Brain.fq  -aligner_name bowtie2  -aligner_bin /user/home/bowtie2 -indexer_bin   ./bowtie2_build  -indices chr1,chr2 -genome /user/home/hg19 -o Brain --ployA

  • GETUTR

    python GETUTR.py -i Brain.bam -o Brain.3UTR -m 10 -r refFlat.txt

  • PHMM

    • Build transcript database
    Rscript buildTranscriptDB_fixed.R  refGene txdb.hg19.refGene.sqlite hg19
    • Select transcripts with 3'utr with length > 600bp
    Rscript apa3utr_fixed.R txdb.hg19.refGene.sqlite 22 long3utr.txt
    • Compute read tag counts in sliding windows
    Rscript apaCount_fixed.R long3utr.txt 22 Brain.bam
    • Fit poisson HMM
    Rscript poissonHMM_fixed long3utr.txt Brain.sorted.cts.rda Brain.csv

  • ChangePoint

    perl change_point.pl -t Brain.bam -c Brain_control.bam -g 3utr.bed -d s -o Brain

    perl change_point.pl -t Brain.bam -c Brain_control.bam -g 3utr.bed -d l -o Brain

  • IsoSCM

    java -Xmx102400m -jar IsoSCM-2.0.11.jar assemble -coverage false -bam Brain.bam -base Brain -s unstranded
java -Xmx102400m -jar IsoSCM-2.0.11.jar assemble -coverage false -bam Brain_control.bam -base Brain_control -s unstranded

    java -Xmx102400m -jar IsoSCM-2.0.11.jar compare -x1 brain.assembly_parameters.xml -x2 brain_control.assembly_parameters.xml -base Brain

  • DaPars

    • Generate region annotation
    python DaPars_Extract_Anno.py -b hg19_refseq_extracted_3UTR.bed -s hg19_4_19_2012_Refseq_id_from_UCSC.txt -o extracted_3UTR.bed
    • Run DaPars
    python DaPars_main.py configure_file  
 configure_file: 
 Annotated_3UTR=hg19_refseq_extracted_3UTR.bed
 Group1_Tophat_aligned_Wig=Brain.bedgraph
 Group2_Tophat_aligned_Wig=Brain_control.bedgraph
 Output_directory=DaPars_Brain/
 Output_result_file=DaPars_Brain
 Num_least_in_group1=1
 Num_least_in_group2=1
 Coverage_cutoff=30
 FDR_cutoff=0.05
 PDUI_cutoff=0.5
 Fold_change_cutoff=0.59

  • APAtrap

    • Identify distal 3' UTR
      For genome having long 3'UTR
    identifyDistal3UTR -i Brain.bedgraph Brain_control.bedgraph -m hg19.genemodel.bed -o Brain.utr.bed  

     For genome having short 3'UTR

    identifyDistal3UTR -i Brain.bedgraph Brain_control.bedgraph -m hg19.genemodel.bed -o Brain.utr.bed -w 50 -e 5000
    • APA site detection
      For genome having long 3'UTR
    predictAPA -i Brain.bedgraph Brain_control.bedgraph -g 2 -n 1 1 -u Brain.utr.bed -o output.txt  

     For genome having short 3'UTR

    predictAPA -i Brain.bedgraph Brain_control.bedgraph -g 2 -n 1 1 -u Brain.utr.bed -o output.txt -a 50
    • APA dynamics detection
    library(deAPA)
deAPA('output.txt', 'de_output.txt', 1, 2, 1, 1, 20)

  • TAPAS

    • APA site detection
    samtools view -b Brain.sorted.bam > Brain.bam
samtools depth Brain.bam > Brain.txt
./APA_sites_detection -ref refFlat.txt -cov Brain.txt -l 50 -o Brain.txt
    • APA dynamics detection
    ./Diff_APA_site_analysis -C1 Brain.txt,Brain_control.txt -C2 UHR.txt,UHR_control.txt -a refFlat.txt -cutoff 70 -type d -o Brain_output.txt

  • EBChangePoint

    perl EBChangePoint.pl -c Brain.bam -t Brain_control.bam -g 3utr.bed  -h1  junctions_brain.bed   -h2 junctions_brain_control.bed

    Junction.bed file for Brain.fq/Brain_control.fa sample, i.e. generated by Tophat