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Manual of ASEproc

Version: 1.0
We developed a new pipeline, ASEproc, which can help user to find allele specific expression site from RNA-seq data. We also built a graph procedure, Graphproc, to help user draw the scatter plot.

The ASEproc's manual and test datset can be downloaded from our FTP site: ftp://treeslab1.genomics.sinica.edu.tw/ASEproc/

1. System requirements

ASEproc runs under the Linux

The R scripts used in ASEproc are developed under 3.3.0

2. Preparation

2.1. External tools

The following three tools are involved in the ASEproc:

   (1) Samtools (http://www.htslib.org/)
   (2) R (https://www.r-project.org/)
   (3) pileup2base (https://github.com/riverlee/pileup2base/blob/master/pileup2base.pl)
2.3. Reference preparation

The genomic sequences was downloaded from the GENCODE website at http://www.gencodegenes.org/. Given the human reference genome (GRCh38.p7, http://www.gencodegenes.org/releases/current.html) as an example:

    Genome sequence FASTA file in GRCh38.p7.genome assembly: GRCh38.p7.genome.fa.gz
2.4. Generating the bam files

The bam files for ASEproc were generated from STAR aligner and Samtools, the users can generate it with the following command:

     > STAR --genomeDir Genome_dir --readFilesIn test_1.fq test_2.fq --runThreadN 10
     > cat <(samtools view -S -H Aligned.out.sam) <(samtools view -S Aligned.out.sam | awk '$5==255{print $0}') > test.sam
     > samtools view -S -b -o test.bam test.sam
     > samtools sort -o test_sort.bam test.bam
     > samtools index test_sort.bam
2.5. Preparing the SNP files

The users must prepare the SNP file.

> chr10	119168031	119168032	+	T	G/T	GT
> chr10	123138743	123138744	+	T	C/T	CT
> chr10	13278235	13278236	+	C	C/T	CT

The column format of SNP file is described as follows:

    (1) Chromosome name 
    (2) 0 base
    (3) 1 base
    (4) strand
    (5) Reference base
    (6) Reference base / Alternative base
    (7) Alleles type

3. Execution of ASEproc and Graphproc

Usage:

 > ./ASE_PROC.sh [Task_name] [Bam_file] [Ref_genome] [SNP_file]
 > ./Graph_PROC.sh [Site_location]

An example:

 > ./ASE_PROC.sh NA06984 NA06984.bam GRCh38.p7.genome.fa NA06984.bed
 > ./Graph_PROC chr1:10000:10001

The test bam files "NA06984.bam", "NA06985.bam" and SNP files "NA06984.bed", "NA06985.bed" can be downloaded from our FTP site:

Note: The Graph_PROC.sh is executed after all ASE_PROC are finished.

4. ASE procedure outputs

The output files are generated after executing the ASE_PROC, these files would be stored in Output folder

> chr1	151760858	151760859	T	G	67	67	1	Biallilic
> chr1	154584186	154584187	G	A	98	66	0.0124621582945403	Partial
> chr1	155017118	155017119	T	C	24	22	0.768082561900943	Biallilic

The column format is described as follows:

(1) Chromosome name 
(2) 0 base
(3) 1 base
(4) Reference base
(5) Alternative base
(6) Reference base reads count
(7) Alternative base reads count
(8) P-value
(9) ASE status