https://bmcmedgenomics.biomedcentral.com/articles/10.1186/s12920-021-01059-x

It is common practice to use Next Generation Sequencing in diagnostics lab to detect SNPs/INDELs. But to detect CNVs diagnostic labs are still using wetlab-based methods, e.g. MLPA (MRC Holland), RNA sequencing, or long-range PCR methods. These methods are expensive, lab-intensive and time consuming. Due to the availability of NGS data it is very relevant to use it to detect also CNVs in diagnostics. Target panels are commonly used in diagnostics labs due to specificity of aims towards checking for variations in specific genes. Our pipeline utilizes NGS data from target panels to detect CNVs for genetic diagnostics.

The following softwares needs to be pre-installed.

• GATK.
• R programming.
• ImageMagick.

git clone https://github.com/ash9nov/Target-panel-based-CNV-detection

GATK's DepthOfCoverage is used on all the BAM files of any NGS run for creating requied input data.

find <path_to_NGS_run_BAM_files> -name "*.bam"| sort > FINAL_BAMs.list

mkdir coverage_report

java -jar GATK/GenomeAnalysisTK.jar -T DepthOfCoverage -R ucsc.hg19.fasta -I FINAL_BAMs.list -o NGS_run -L Target_panel.bed

java -jar GATK/GenomeAnalysisTK.jar DepthOfCoverage -R ucsc.hg19.fasta -I FINAL_BAMs.list -O NGS_run -L Target_panel.bed

per_locus_coverage file (consisting of nucleotide level coverage of each sample in RUN) and run_summary file (consisting of mean coverage of each sample in RUN) are used by pipeline for the analysis

To increase resolution each target region is divided into overlapping sub-regions in a sliding window approach (as shown in figure below), forming the template for a window-based representation of each target region. This approach is called the Target Region based Sliding Windows (TRSW) approach, or just sliding windows. This also helps in detecting CNVs occurring in smaller sub-regions, e.g., part of an exon. Selection of window size is based on length of sequencing reads and the required resolution of CNV predictions.

step0_R_code_for_breaking_target_regions_on_fixed_window_size.r

Here default length of window is 75 nucleotide, sliding length is 10.

Pipeline generates static pools from normal samples (with no CNVs), sorted according to coverage depth. The pipeline can then select a pool of samples that matches the coverage depth of the query sample and use this to estimate expected coverage depth (without any CNVs) for a region of interest.

The figure below shows the steps of creating static pools from normal samples. In step-1 normal samples are selected from available NGS runs and get listed in order of increasing coverage depth. In step-2 the coverage depth is calculated for each window across each sample. In step-3 the list of selected normal samples is divided into different pools of size K, where Pool-1 consists of the first K samples, followed by the next pool consisting of the next K samples after skipping the first sample of the previous pool. In step-4 the mean TRSW of each pool is calculated.

sh Steps_for_generating_static_pooling.sh

INPUT : List of NGS runs (provided as text file) consisting of samples to be used in static pools, and pool size.

OUTPUTS :

This script uses RUN.sample_summary and per_locus_coverage files (output from GATK's DepthOfCoverage) from provided List_of_runs .

A. Creates sorted list of samples with coverage information.

B. Creates sample's sliding window level coverage via script Step1.1_code_to_generate_Sample.Sliding_Window_file.sh

C. Splits the list of samples in different pools with given pool size "K".

D. Generating Info_Table_of_Pools which contains mean coverage of each pool via script NGS_CNV_code/step2_R_code_for_SlidingWindow_MeanDepth_calculation.r

E. Generating Pool_TRSW_mean_depth. via running the script Step1.2_R_code_for_POOL_MEAN_TRSW_calculation.r

For a given query sample the coverage depth is first calculated for each sliding window. A static pool is then chosen from the set of static pools where mean coverage depth of the selected pool is closest to coverage depth of the sample. The coverage depth for each window of the query sample is compared against mean coverage depth of each corresponding window of the selected pool. This ratio is converted to log2 scale to calculate the final CNV score, i.e., log copy number ratio score (logCNR score) for that window.

Below is exmple plot output of NF1-ex48 deletion (included info Run and static pools avg_cvg for easy comparision)

The figure below shows the general workflow for CNV calculation. In step-1 sliding window level (TRSW) coverage for the query sample is calculated. In step-2 a static pool is selected (from the list of pools) based on its mean coverage depth similarity to the query sample's coverage depth. In step-3 "log-copy-number-ratio" is calculated for for each sliding window of query sample, which gets gene annotated in step-4. This is the final output of the pipeline.

Pipeline workflow

sh step1_code_for_running_CNV_analysis_parallel_NGS_pipeline_integration.sh

This script will do the follwoing calculations:

A. Calculates mean coverage depth at sliding window level for query samples using the R script step2_R_code_for_SlidingWindow_MeanDepth_calculation.r

B. Runs this script step1_shell_code_for_running_CNV_analysis_with_STSTIC_pooling.sh which calculates following

B.1. Selects the most suitable pool among available pools using R script step2_R_code_for_pool_selection.r

B.2. Calculates logCopyNumberRatio for all sliding windows of query sample w.r.t. selected static pool using script step3_R_code_for_logCopyNumberRation_calculation_with_STATIC_pooling.r

B.3. Plots the logCopyNumberRatio score calculated in previous step using script step4_R_code_for_plotting_sample_for_each_individual_gene_static_pooling.r

B.4. Calculates the % coverage daviation for query sample vs selected pool using script step5_R_code_for_quality_control_in_STATIC_pooling.r

C. Concatenates all the plots for different genes one under each other in a single image using script step_FINAL_code_for_combining_all_runwise_and_static_pooling_plots_from_all_samples_for_each_gene.sh