CoverageAnalysisTool

Outputs a list of coding regions that are uncovered above a given threshold

Overview

• COAT is a Python2 program developed in 2016 which aims to automatically find bad quality region of coding sequences in a set of individual exome sequencing data.

• It is a module integrated to the EXome Analysis and Mining.

• It includes a graphical interface.

• The output of this program is a spreadsheet with all the coding regions which are uncovered. Supplementary annotation can be used for further analysis. Additional files can be generated using --cover_output argument and/or --track_no_cover_UCSC_output.

Input

BAM files

BAM is a binary file format. SAM and BAM files contain the same information. These files contain mapped reads sequence from Next Generation Sequencing.

Installing COAT on LINUX

To use COAT, the following programs are necessary :

• python 2.6
• PyQt4
• samtools 1.3

The following files are necessary :

• BAM files folder
• reference sequence annotation file

Programming language Python

Python (version 2.6.5 to version 2.7.8). To see which version of Python you have installed, open a command prompt and run:

python --version

If you don’t have Python, type the following command to install python version 2.x:

sudo apt-get install python 2.6

GUI toolkit PyQt4

If you want to use COAT graphical interface, you will need to install the python module PyQt4

apt-cache search pyqt
sudo apt-get install python-qt4

Samtools

To run COAT, you must have installed samtools 1.3. If you have a previous version of samtools, COAT will not work, so download and install samtools 1.3. Unzip the downloaded file. Go into the newly created directory and compile the code by typing "make".

tar -xvjf samtools-1.3.tar.bz2
cd samtools-1.3/
make

BAM

Store your BAM files into a dedicated folder. Prefix of BAM files name is used as subject identifier (see command-line argument --subject_list).

Samtools folder must be specified (see command-line argument --samtoolsPath) BAM files must be indexed. To generate Index of BAM files, use samtools line-commands:

samtools sort -T /tmp/aln.sorted -o aln.sorted.bam aln.bam
samtools index aln.sorted.bam

Reference sequence annotation file

Reference annotated sequence is needed. This file contains gene annotation of the genome reference you want to use. You can get file for h19 reference:

• http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refFlat.txt.gz

Command-line Arguments

This table summarizes the command-line arguments which are using by COAT.

complete flag argument	short flag	Default value	Summary
--output	-o	stdout	Output folder path. Will overwrite contents if file exists
--position	-p	NA	Interval positions in chromosome of the reference sequence FORMAT: chr:start-end
--geneName	-g	NA	Select region of the gene. FORMAT: name of the gene according to reference
--cds_regions	-cds	TRUE	regions of exons which are not into UTR in the reference sequence
--add_utr_regions	-add_utr	FALSE	Add UTR to the selected region
--add_intron_regions	-add_intron	FALSE	Add intron regions to the selected region
--bamsPath	-bams	NA	BAM files folder
--subject_list	-subject	NA	Coma-separated list of prefix BAM files name(s) of selected subject in BAM files repertory
--coverage_threshold	-cov	5	The minimum coverage to be actually considered as covered
--mapQuality_threshold	-mapqual	20	The minimum allowable mapping quality score to be counted for coverage
--baseQuality_trehshold	-basequal	0	The minimum allowable base quality score to be counted for coverage
--cover_output	-covout	FALSE	Output as a supplementary file the list of covered regions with annotation
--track_no_cover_UCSC_output	-track	FALSE	Output UCSC Genome Brower custom track which contains uncovered regions
--refPath	-ref	NA	Annotation file for reference e.g. hg19
--samtoolsPath	-samtools	NA	Samtools 1.3 folder path

Example of command

python2 coverageAnalysis.py \
-gene GLIS3 \
-cds \
-add_utr \
-bams /storage/bams/ \
-subject family1_1,family1_3 \
-ref refFlat_hg19.txt \
-cov 15 \
-o coverage_check_results/

Output

List of uncovered/covered coding regions with annotation

Chrom	Uncovered_start	Uncovered_end	subject	gene	transcript	Exon_numb	Uncovered_length
Chr9	3937027	3937120	Family1_1	GLIS3	NM_001042413	5	94
Chr9	3937027	3937074	Family1_3	GLIS3	NM_001042413	5	48

Each row is an uncovered region.

• Chrom: chromosome.
• Uncovered_start: the reference start position of an uncovered region.
• Uncovered_end: the reference end position of an uncovered region.
• subject: the sequenced subject identifier.
• gene: name of the gene including the uncovered region.
• transcript: name of the transcript of the gene including the uncovered region.
• exon_numb: numb of the exon of the transcript of the gene including the uncovered region. This field can optionally mention UTR5’, UTR3’ and intron region.
• uncovered_length: number of nucleotides in the uncovered region.

UCSC Genome browser custom track of uncovered region

Visualising uncovered region on gene GLIS3 of the 2 subjects from family 1 with UCSC Genome Browser. Annotation from REFSEQ have been added. In red, uncovered region.

Interface

To run in graphical mode the program :

python2 Interface/coat_HCI.pyw

This command will display the following interface :

---

1. Advanced settings, save settings and reset

Click on Advanced settings to access advanced settings section.

• bam files folder: path of the folder in which your BAM files and BAM.bai files are stored.
• samtools folder: path of the folder in which samtools 1.3 is stored.
• Hg reference table file: path of the reference annotated sequence.
• Results folder: output folder path. Will overwrite contents if file exists.

Save settings will save all your settings for the next time you will use COAT. Reset gives the default value for all settings.

2. Select subject

Fill the fields with your coma-separated list of prefix BAM files name(s) of selected subject in BAM files folder.

3. Select region

Select the region to check by specify coordinates else or gene name. Coordinates refers to an interval positions in chromosome of the reference sequence (FORMAT: chr:start-end). By default, coverage check is running on CDS region. But you can add UTR and intron region by toggle the corresponding option.

4. Select coverage thresholds

3 thresholds are availables:

• the minimum allowable coverage depth to be considered covered (default value = 5);
• the minimum allowable mapping quality score to be counted for coverage (default value = 20);
• the minimum allowable base quality score to be counted for coverage (default value = 0).

5. Generate additional files

By default, output is a list of uncovered coding regions with annotation. You can add by toggle the corresponding option: a list of covered regions with annotation; an UCSC custom track which contains uncovered regions.

6. Launch and quit

When all your parameters are selected, click on launch coverage check to run COAT.