Ribosomal DNA Copy Number calculation

The pipeline is constructed in a way to consider data from the TCGA data portal.

Once the data are downloaded using gdc-portal, you need to download the sample sheet file that contains information about the samples. The sample sheet file would be used by the function convertNames.pl to change the files name, get the sample's ID (see column Sample Type) and the UUID (see column File ID).

It is important to note that all the downloaded files are in separate folders and named according the UUID column. More, in each folded there is a bam file named according the file name column.

Basic Usage

_{The pipline is build by Meng Wang (2016) at harvard T.H.chan School of public health (Lemos Lab) and contributor(A. Bedrat).}

Features

• Written in perl and shell.
• No installation necessary.
• To use, you need to change the paths of the run file.
• Works on Mac and Linux (never tested on Windows).

Needed files

Make sure to Download (a test data set is already provided in /DATA) and change the path of all the files bellow:

• 45S and 5S consensus sequences U13369.1 (see DATA/rDNA.fa where both 45S and 5S are in one file).
• rDNA components location (see DATA/Loci.bed)
• The sample sheet (see introduction). (The first ligne "title ligne" should be deleted)
• Fastq or bam files. If you use bam files, the tool will extract the fastq sequences aligned on the human genome.
• Human GTF File Format GRCh38.p12.gtf
• Single Exons and Introns copy used as genome background [Material and methods].(https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006994#sec011) (see DATA/Exon_Intron_6chr.bed)
• Gene id and names ensembl/Biomart(see DATA/Ensembl2Symbol-GrH38.txt).
• Gene-level copy number values for tumor samples FireBrowse/SNP6 Copy number analysis (GISTIC2) (see DATA/all_data_by_gene_PRAD.txt).

Used tools

• bamUtil
• BWA
• samtools
• GATK
• perl

Usage

The pipeline runs on two steps:

Alignenent and depth calculation

To run the first step:

$ cd PATH/TO/RUN1.sh
$ RUN1.sh

This step will:

• Extract and map reads onto the rDNA consensus sequences (rDNA.fa).
• Estimate the rDNA and the exon/intron background depth.
• Calling variants.

After this step, X files are generated. They are used through the second step.

Copy number calculation:

To run the second step:

#YOU NEED TO CHANGE THE:
#UUID =>Line 4
#Specify the path of the results output folders (line 9)
#THE PATH OF THE 14 FILES (from line 20 to 35)
#Know you can run 

$ cd PATH/TO/RUN2.sh
$ RUN2.sh

This step will:

• Calculate the depth of the rDNA componenents (18S, 5.8S, 28S and 5S).
• Calculate the backgroud depth for tumor and normal samples.
• Calculate the exons and intron mean Background depth.
• Calculate the rDNA copy number.

Results:

The rDNA copy number is summerized in the file rDNA_CN/CN-sampleID.txt

rDNA_subunit	Background_Depth	rDNA_Depth	rDNA_CN	Type
18mS	24.22	13.8352214212152	0.571231272552238	exon
18mS	3.47	13.8352214212152	3.98709551043666	intron
18mS	13.85	13.8352214212152	0.999293710452524	meanEX-IN

Contributing

Bug Reports & Feature Requests

Please use the issue tracker to report any bugs or file feature requests.