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Comprehensive genome-wide visualization of absolute copy number and copy neutral variations

License: GNU General Public License v3.0

Dockerfile 0.30% Python 94.72% R 4.97%

acnviewer's Introduction

aCNViewer

comprehensive genome-wide visualization of absolute copy number and copy neutral variations

Contact: Victor Renault / Alexandre How-Kit ([email protected])

aCNViewer (Absolute CNV Viewer) is a tool which allows the visualization of absolute CNVs and cn-LOH across a group of cancer samples. aCNViewer proposes three graphical representations : dendrograms, bidimensional heatmaps allowing the visualization of chromosomal regions sharing similar abnormality patterns and quantitative stacked histograms facilitating the identification of recurrent absolute CNVs and cn-LOH. aCNViewer include a complete pipeline allowing the processing of raw data from SNP array (in tumor-only or paired tumor / normal mode) and whole exome/genome sequencing experiments (in paired tumor / normal mode only) using respectively ASCAT and Sequenza algorithms to generate absolute CNV and cn-LOH data used for the graphical outputs.

Table of contents

Installation

Docker installation

The easiest way to install aCNViewer is to install the Docker application (supports multi-threading but not computer clusters which are better suited for processing NGS bams): docker pull fjdceph/acnviewer

aCNViewer docker image requires about 20GB of space to install so if you run into an error while pulling the image locally, you probably need to change the location of docker images from /var/lib/docker/ to a location with more space and try again.

Installation from source

aCNViewer can also be installed from its source by:

  1. downloading aCNViewer's data (includes test data sets and most of the third-party softwares listed in the dependencies section)
  2. installing the dependencies listed below.
  3. downloading the github source code from this page: git clone https://github.com/FJD-CEPH/aCNViewer

Installation validation

Once aCNViewer is installed, you can run unit tests in order to check that everything is fine.

Dependencies:

Most of the dependencies (except R and python), along with test data sets, are packaged in the archive aCNViewer_DATA.tar.gz in aCNViewer_DATA/bin. You can find more details below:

  • APT (Affymetrix Power Tools) if you plan to process raw Affymetrix SNP arrays (to uncompress into BIN_DIR)

  • a recent version of R (version ≥ 3.2) with ggplot2 installed for generating the different graphical outputs:

    • ASCAT (will be automatically installed if not already installed) if you are analyzing raw SNP array data
    • Sequenza (will be automatically installed if not already installed) if you are analyzing paired (tumor / normal) bams
    • plotrix for plotting dendrograms (will be automatically installed if not already installed)
    • gplots
    • RColorBrewer

  • samtools if you are analyzing paired (tumor / normal) bams. As Sequenza does not support newer mpileup file formats produced by more recent versions of samtools, use a version prior to Sequenza release date (2015-10-10): samtools version 0.1.19 for example is compatible.

  • tQN if you plan to process raw Illumina SNP arrays (to uncompress into BIN_DIR) and run tQN normalisation. If the cluster file for the Illumina SNP array you plan to analyze is not in the tQN lib folder, you can download additional cluster files from here

  • GISTIC if you want to have an advanced statistical way to prioritize regions of interest. Create a folder named GISTIC_VERSION in BIN_DIR and uncompress the GISTIC archive into it. Follow the instructions listed in INSTALL.txt at the root of the GISTIC folder in order to install MATLAB Component Runtime required by GISTIC and set the associated environment variables (LD_LIBRARY_PATH and XAPPLRESDIR).

  • Python with version ≥ 2.7

Overview:

Overview of aCNViewer:

Tutorial

The results of all the examples below can be found in aCNViewer_DATA/allTests in their respective target folder. All examples of this tutorial are implemented as unit tests and can be run at once using: DOCKER_OR_PYTHON -P testAll -t TARGET_DIR [--fastTest 0 --smallMem 0 --runGISTIC 0].

If --fastTest is set to 1, only tests which run in a reasonable amount of time will be run (all tests except Illumina SNP array, paired bams with Sequenza, GISTIC and Affymetrix SNP arrays from CEL files). If --runGistic is 1, GISTIC will be tested and if --smallMem is set 1, GISTIC will run in small memory mode and will only require about 10GB of RAM vs 50GB of RAM at the expense of a longer running time.

Glossary:

Let's call:

  • aCNViewer_DATA the location where the test data set aCNViewer_DATA.tar.gz has been uncompressed into

  • BIN_DIR the folder containing all third-party softwares located in aCNViewer_DATA/bin. Here is the list of files and folders that should be in BIN_DIR:

    • apt-*: Affymetrix Power Tools binaries
    • ascat: contains ASCAT file for GC correction (this folder is automatically created by aCNViewer and GC files are automatically downloaded)
      • GC_Affy250k.txt
      • GC_AffySNP6_102015.txt
      • gc_done
      • GC_Illumina660k.txt
      • GC_IlluminaOmniexpress.txt
    • GISTIC*: installation folder of GISTIC
    • PennCNV: contains PennCNV-Affy protocols and helper scripts (will be automatically created by aCNViewer from gw6.tar.gz)
    • samtools*: installation folder of samtools
    • tQN*: installation folder of tQN
    • ../genomes: folder located in the parent folder of BIN_DIR with one folder per genomic build. Each genomic build folder BUILD should contain at least:
      • one file named BUILD.centro.txt with centromere positions for each chromosome of the genomic build (can be generated using curl -s "http://hgdownload.cse.ucsc\ .edu/goldenPath/BUILD/database/cytoBand.txt.gz" | gunzip -c | grep acen > BUILD.centro.txt)
      • a tab-delimited file named BUILD.chrom.sizes with 2 columns respectively chromosome name and chromosome length (can be downloaded from UCSC Genome browser)
      • optionnaly, a reference fasta file (with one of the extension .fa, .fa.gz, .fasta or .fasta.gz) if you plan to use Sequenza

  • DOCKER_OR_PYTHON refers to the fact that docker run fjdceph/acnviewer or python aCNViewer/code/aCNViewer.py can be used as a prefix to run aCNViewer depending on the chosen installation method.

Requirements:

Download the test data set aCNViewer_DATA.tar.gz (~5GB and ~20GB uncompressed). In terms of computing resources: if you plan to:

  • run Sequenza on paired bam files, an access to a computer cluster is highly recommended as even though aCNViewer will be able to process your data in multi-threading mode, it may take quite a long time depending on the number of sample pairs to analyze
  • run GISTIC in order to have a robust statistical way to prioritize recurrent regions of interest, a machine with at least 50GB of RAM is necessary with --smallMem 0 and 10GB with --smallMem 1 (this option will make GISTIC run substantially longer)

Processing SNP array data

Affymetrix

TestAffyAscat

Generate all available plots from ASCAT segment files using base resolution for the quantitative histograms and using a window size of 2Mbp for the other plots:

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt

quantitative stacked histogram example:

Histogram of heterozygous / homozygous CNVs:

Here are other typical plots you may be interested in:

Customize colors:

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_RCOLOR --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --rColorFile aCNViewer_DATA/rColor.txt

Quantitative histogram with GISTIC results:

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_GISTIC --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --runGISTIC 1

If you have trouble running this example (in particular if your machine freezes or you get the message "Killed" in the "_gistic.txt.err" file), it may be due to a lack of resources in the machine you are using. In that case, please add the following option to the command above --smallMem 1 so that GISTIC runs in compressed memory mode. You can view the GISTIC results with significant broad events and significant focal events.

Heatmap of relative copy number values only for the clinical feature BCLC stage with the chromosome legend position set at 0,.55 i.e. at the left-most of the graph and at 55% on the y axis and the group legend position set at .9,1.05 (basically at the top right corner):

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_HEATMAP1 --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --plotAll 0 --heatmap 1 --dendrogram 0 -G "BCLC stage" --chrLegendPos 0,.55 --groupLegendPos .9,1.05 --useRelativeCopyNbForClustering 1

Heatmap of relative copy number values using the clinical feature BCLC stage:

Heatmap with regions ordered by genomic positions (only clustering on samples):

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_HEATMAP_GENPOS --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --plotAll 0 --heatmap 1 --dendrogram 0 -G "BCLC stage" --chrLegendPos 0,.55 --groupLegendPos .9,1.05 --useRelativeCopyNbForClustering 1 --keepGenomicPosForHistogram 1

Heatmap of relative copy number values with regions ordered by genomic positions using the clinical feature BCLC stage:

Heatmap with copy number values:

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_HEATMAP2 --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --plotAll 0 --heatmap 1 --dendrogram 0 -G "BCLC stage" --chrLegendPos 0,.55 --groupLegendPos .9,1.05

Heatmap of copy number values using the clinical feature BCLC stage:

Dendrogram with copy number values:

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_DENDRO --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --plotAll 0 --heatmap 0 --dendrogram 1 -G "BCLC stage" -u 1

Dendrogram of copy number values using the clinical feature BCLC stage:

Customize output formats:

  • all outputs set to pdf: DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_PDF --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --outputFormat pdf

  • all output set to jpg: DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_PDF --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --outputFormat jpg

  • heatmaps set to bmp, histograms to tiff and dendrograms to pdf with the R plot parameters width=10,height=8: -f aCNViewer_DATA/snpArrays250k_sty/GSE9845_lrr_baf.segments.txt -t TEST_AFFY_OTHER_OUT --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --sampleFile aCNViewer_DATA/snpArrays250k_sty/GSE9845_clinical_info2.txt --outputFormat "heat:bmp;hist:tiff;dend:pdf(width=10,height=8)"

==Here is the full command:==

DOCKER_OR_PYTHON -f ASCAT_SEGMENT_FILE --refBuild REF_BUILD -b BIN_DIR [--histogram HISTOGRAM --lohToPlot LOH_TO_PLOT --useFullResolutionForHist USE_FULL_RESOLUTION_FOR_HIST] [-c CHR_SIZE_FILE -t OUTPUT_DIR -C CENTROMERE_FILE -w WINDOW_SIZE --sampleFile SAMPLE_FILE -G PHENOTYPIC_COLUMN_NAME --rColorFile RCOLOR_FILE --plotAll PLOT_ALL --outputFormat OUTPUT_FORMAT --ploidyFile PLOIDY_FILE --sampleToProcessList SAMPLE_TO_PROCESS_LIST --sampleToExcludeList SAMPLE_TO_EXCLUDE_LIST --sampleAliasFile SAMPLE_ALIAS_FILE] [--heatmap HEATMAP --labRow LAB_ROW --labCol LAB_COL --cexCol CEX_COL --cexRow CEX_ROW --height HEIGHT --width WIDTH --margins MARGINS --hclust HCLUST --groupLegendPos GROUP_LEGEND_POS --chrLegendPos CHR_LEGEND_POS --useRelativeCopyNbForClustering USE_RELATIVE_COPY_NB_FOR_CLUSTERING --keepGenomicPosForHistogram KEEP_GENOMIC_POS] [--dendrogram DENDROGRAM --useShape USE_SHAPE] [--runGISTIC RUN_GISTIC --geneGistic GENE_GISTIC --smallMem SMALL_MEM --broad BROAD --brLen BR_LEN --conf CONF --armPeel ARM_PEEL --saveGene SAVE_GENE --gcm GCM]
where:

  • ASCAT_SEGMENT_FILE: ASCAT segment file (ascat.output$segments obtained by running ascat.runAscat) with the following columns:
    • sample
    • chr
    • startpos
    • endpos
    • nMajor
    • nMinor
  • REF_BUILD: the genome build used to generate the CNV segments (hg18 and hg19 are currently supported. If you want to add another build BUILD, please add a folder in BUILD in aCNViewer_DATA/genomes containing at least a tab-delimited file named BUILD.chrom.sizes with each chromosome name and length and a tab-delimited file named BUILD.centro.txt with the centromere positions by chr [this file can be generated using curl -s "http://hgdownload.cse.ucsc.edu/goldenPath/BUILD/database/cytoBand.txt.gz" | gunzip -c | grep acen > centro_build.txt])

The following options are general plotting options:

  • CHR_SIZE_FILE: a tab-delimited file with 2 columns respectively chromosome name and chromosome length. When REF_BUILD is set, CHR_SIZE_FILE is automatically set to aCNViewer_DATA/genomes/REF_BUILD.chrom.sizes.
  • CENTROMERE_FILE: file giving the centromere bounds. Can be generated using curl -s "http://hgdownload.cse.ucsc.edu/goldenPath/BUILD/database/cytoBand.txt.gz" | gunzip -c | grep acen > centro_build.txt. When REF_BUILD is set, CENTROMERE_FILE is automatically set to aCNViewer_DATA/genomes/REF_BUILD.centro.txt.
  • WINDOW_SIZE: segment size in bp. Please note that alternatively, -p PERCENTAGE can be used instead of -w WINDOW_SIZE in order to set the segment size in percentage of chromosome length where PERCENTAGE is a floating number between 0 and 100. If WINDOW_SIZE and PERCENTAGE are null then WINDOW_SIZE is set to 2Mb by default.
  • SAMPLE_FILE: a tab-delimited file that should contain a column named Sample with the name of each sample and at least another column with the phenotypic / clinical feature. This file can contain a sampleAlias which will be used as the official sample id if provided.
  • PHENOTYPIC_COLUMN_NAME is optional and refers to the name of the column of the phenotypic / clinical feature of interest in SAMPLE_FILE. If you omit this parameter, one plot per feature defined in SAMPLE_FILE will be generated.
  • RCOLOR_FILE: file allowing to customize graph colors: colors for histograms can be overriden using a section named "[histogram]" which should contain exactly 10 colors [one per line] corresponding to CNV values in the following order: "≤ -4", "-3", "-2", "-1", "1", "2", "3", "4", "5", "≥ 6"). Histogram colors for heterozygous / homozygous CNVs can be changed using the section "[heteroHomo]" which should contain 6 colors corresponding to the values "-Hom", "-Het", "=Hom", "=Het", "+Hom", "+Het". Colors for dendrograms can be redefined using the section "[group]" which should contain at least the same number of colors than the number of distinct values for the phenotypic / clinical feature of interest. Colors for heatmaps are customizable using the section "[chr]" and should contain 22 colors corresponding to chromosomes 1 to 22], the section "[group]" (the same as previously seen for dendrograms) and the section "[heatmap]" which should contain 10 colors (one per line) corresponding to CNV values in the following order: "0", "1", "2", "3", "4", "5", "6", "7", "8", "≥ 9". An example can be found here.
  • PLOT_ALL: specify whether all available plots should be generated. The default value is 1.
  • OUTPUT_FORMAT: allow to customize output formats for the different types of available plots (histograms, heatmaps and dendrograms). Examples of use can be found above. The default value is hist:png(width=4000,height=1800,res=300);hetHom:png(width=4000,height=1800,res=300);dend:png(width=4000,height=2200,res=300);heat:pdf(width=10,height=12).
  • PLOIDY_FILE: custom ploidy values for each sample. Can either be a tab-delimited file with at least 2 columns: "sample" and "ploidy" or an integer which will set the same ploidy to all samples. By default, the ploidy is calculated using the CNV file segmented in fragments of 10% of chromosomal length and its value will be the most represented CNV value for each sample.
  • SAMPLE_TO_PROCESS_LIST: comma-separated string or file with one sample per line used to restrict the list of samples to process by aCNViewer.
  • SAMPLE_TO_EXCLUDE_LIST: comma-separated string or file with one sample per line used to exclude a list of samples from analyses.
  • SAMPLE_ALIAS_FILE: optional parameter used to change the sample name to a preferred sample name. It is a tab-delimited file with 2 columns: one for the sample name and a second one with the preferred sample name.

The following options are histogram specific:

  • HISTOGRAM: specify whether an histogram should be generated. The default value is 0 but its value is overriden to 1 when option --plotAll 1 is set.
  • LOH_TO_PLOT: histogram option for LOH plotting. Values should be one of "cn-LOH" for plotting cn-LOH only, "LOH" for LOH only, "both" for cn-LOH and LOH or "none" to disable this feature. The default value is "cn-LOH".
  • USE_FULL_RESOLUTION_FOR_HIST: tell whether to plot histogram using full resolution i.e. CNVs are not segmented according to a user-defined length through windowing approach. The default value is 1. If 0, the resolution of the plot will be given by either WINDOW_SIZE or PERCENTAGE.

The following options are GISTIC options (more details can be found here):

  • RUN_GISTIC: specify whether to run GISTIC in order to have a statistical way to prioritize regions of interest. The default value is 0.
  • GENE_GISTIC: tell whether gene GISTIC algorithm should be used to calculate the significance of deletions at a gene level instead of a marker level. The default value is 1.
  • SMALL_MEM: tell GISTIC whether to use memory compression at the cost of a longer runtime. The default value is 0.
  • BROAD: tell GISTIC to run the broad-level analysis as well. The default value is 1.
  • BR_LEN: set GISTIC's broad_len_cutoff. The default value is 0.5.
  • CONF: set the confidence level used to calculate the region containing a driver. The default value is 0.9.
  • ARM_PEEL: set GISTIC's arm_peeloff. The default value is 1.
  • SAVE_GENE: tell GISTIC whether to save gene tables. The default value is 1.
  • GCM: set GISTIC's gene_collapse_method. The default value is extreme.

The following options are mainly specific to heatmaps while a few are related to dendrograms:

  • HEATMAP is an optional parameter used only if PLOT_ALL is set to 0 to tell whether to plot heatmaps or not. The default value is 1
  • LAB_ROW is an optional parameter telling whether heatmap's row names (chromosomal regions) should be shown. The default value is 0
  • LAB_COL is an optional parameter telling whether heatmap's column names (sample names) should be shown. The default value is 1
  • CEX_COL is an optional parameter setting cexCol for heatmaps. The default value is 0.7. See R heatmap.2 documentation for more details
  • CEX_ROW is an optional parameter setting cexRow for heatmaps. The default value is 0.45. See R heatmap.2 documentation for more details
  • HEIGHT is an optional parameter setting height for heatmaps. The default value is 12. for heatmaps.
  • WIDTH is an optional parameter setting width for heatmaps. The default value is 10. See R heatmap.2 documentation for more details
  • MARGINS is an optional parameter setting margins as a comma-separated string for heatmaps. The default value is 5,5. See R heatmap.2 documentation for more details
  • HCLUST is an optional parameter setting hclust method for heatmaps / dendrograms. See R heatmap.2 documentation for more details
  • GROUP_LEGEND_POS is an optional parameter setting the phenotypic / clinical feature legend's position within the heatmap. The default value is topright and can be changed to coordinates (for example 0.1,0.5 which will put the legend at 10% of the total width of the graph on the x axis and 50% of the total height of the graph on the y axis i.e. in the middle of the y axis) or in R specified logical location (top, bottom, left, right, etc)
  • CHR_LEGEND_POS is an optional parameter setting the chromosome legend's position within the heatmap. The default value is bottomleft and can be changed to coordinates (for example 0.1,0.5 which will put the legend at 10% of the total width of the graph on the x axis and 50% of the total height of the graph on the y axis i.e. in the middle of the y axis) or in R specified logical location (top, bottom, left, right, etc)
  • RCOLOR_FILE
  • USE_RELATIVE_COPY_NB_FOR_CLUSTERING is an optional parameter specifying whether the CNV matrix used for the heatmap should be relative copy number values or not. The default value is 0. If PLOT_ALL is 1 then plots for both values of USE_RELATIVE_COPY_NB_FOR_CLUSTERING will be generated.
  • KEEP_GENOMIC_POS is optional and will keep the segmented genome in its original position if set to 1 and not cluster segments according to sample CNV patterns (the default value is 0).
  • DENDROGRAM is an optional dendrogram parameter used only if PLOT_ALL is set to 0 to tell whether to plot dendrograms or not. The default value is 1
  • USE_SHAPE is an optional dendrogram parameter and if set to 1 (default value) will replace sample labels with colored shapes in the leaves of the dendrogram(s).
TestAffyCel

Generate a quantitative stacked histogram from CEL files (subset of data of hepatocellular carcinomas with hepatitis C virus etiology used in Chiang et al. Cancer Res, 2008) with a window size of 2Mbp:

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrays250k_sty/ -t TEST_AFFY_CEL --refBuild hg18 -w 2000000 -b aCNViewer_DATA/bin --platform Affy250k_sty -l aCNViewer_DATA/snpArrays250k_sty/LibFiles/ [--useCustomPloidies USE_CUSTOM_PLOIDIES]

If ASCAT is not installed (i.e you are not using the docker application) and if you want to install it into a custom R library folder, please add the following option to the previous command line: --rLibDir RLIB.

==Here is the full command:==

DOCKER_OR_PYTHON -f CEL_DIR --refBuild REF_BUILD -t OUTPUT_DIR -b BIN_DIR --platform AFFY_PLATFORM -l AFFY_LIB_DIR [--gw6Dir GW6_DIR] [--gcFile ASCAT_GC_FILE] [GENERAL_PLOT_OPTIONS] [HISTOGRAM_OPTIONS] [GISTIC_OPTIONS] [HEATMAP_DENDRO_OPTIONS]
where:

  • CEL_DIR is the folder containing ".cel" ou ".cel.gz" files
  • AFFY_PLATFORM: name of ASCAT supported Affymetrix platform with a GC content file available ("Affy250k_sty", "Affy250k_nsp", "Affy500k" or "AffySNP6"). Please refer to ASCAT website for more details
  • AFFY_LIB_DIR: Affymetrix library file downloadable from Affymetrix website
  • GW6_DIR is optional and refers to the folder where gw6.tar.gz has been uncompressed into. This archive contains different programs and files necessary to process Affymetrix SNP array and has been uncompressed into aCNViewer_DATA/bin/PennCNV/gw6/ (default value).
  • ASCAT_GC_FILE: GC content file necessary for ASCAT GC correction when analyzing SNP array data. This parameter is optional as its value will be automatically deduced from the value of AFFY_PLATFORM. Please check ASCAT website for available GC content files. It is also possible to create custom GC file.
  • USE_CUSTOM_PLOIDIES: specify whether ploidies should be calculated using our custom algorithm (use a window of 10% of chromosomal length and set the ploidy to the most frequent CNV value for each sample) or use ploidies calculated by ASCAT/Sequenza. The default value is 1.

Illumina

TestIllu660k

Generate a quantitative stacked histogram from raw Illumina data from non-Hodgkin lymphoma patients used in Yang F et al. PLoS One 2014 with a window size of 2Mbp:

DOCKER_OR_PYTHON -f aCNViewer_DATA/snpArrayIllu660k/GSE47357_Matrix_signal_660w.txt.gz -t TEST_ILLU --refBuild hg19 -w 2000000 -b aCNViewer_DATA/bin --probeFile aCNViewer_DATA/snpArrayIllu660k/Human660W-Quad_v1_H_SNPlist.txt --platform Illumina660k --beadchip "human660w-quad"

==Here is the full command:==

DOCKER_OR_PYTHON -f ILLU_FILES --refBuild REF_BUILD -b BIN_DIR [--sampleList SAMPLE_TO_PROCESS_FILE] --probeFile PROBE_POS_FILE --platform ILLUMINA_PLATFORM [--beadchip BEADCHIP] [-g ASCAT_GC_FILE] [-N NORMALIZE] [GENERAL_PLOT_OPTIONS] [HISTOGRAM_OPTIONS] [GISTIC_OPTIONS] [HEATMAP_DENDRO_OPTIONS]
where:

  • ILLU_FILES can either be the list of Illumina final report files to process specified either as a comma-separated string with all the report files to process or as a directory containing these files. Each Illumina final report file should contain at least the following columns:

    • SNP Name
    • Sample ID
    • Log R Ratio
    • B Allele Freq

    Alternatively, it can be the raw Illumina files with at least the following columns:

    • ID
    • SAMPLE1.X
    • SAMPLE1.Y
    • ...
    • SAMPLEn.X
    • SAMPLEn.Y

  • CHR_SIZE_FILE

  • CENTROMERE_FILE

  • WINDOW_SIZE

  • PROBE_POS_FILE: file listing the probes used on the SNP array with their genomic position. The file is tab-delimited with the following columns:

    • Name
    • Chr
    • MapInfo or Position

  • ILLUMINA_PLATFORM: name of ASCAT supported Illumina platform with a GC content file available ("Illumina660k" or "HumanOmniExpress12"). Please refer to ASCAT website for more details

  • BEADCHIP:

  • ASCAT_GC_FILE

  • NORMALIZE: Turn on / off tQN normalization. The default value is 1.

  • SAMPLE_TO_PROCESS_FILE: optional, used to specify list of samples to process in one of the following formats:

    • a comma-separated string listing all the samples to process
    • the name of text file with one line per sample to process
    • the name of a Python dump file with the extension ".pyDump"

  • LOH_TO_PLOT

NGS

Sequenza is used to process NGS paired (tumor / normal) bams and produce CNV segments. These segments are then used by aCNViewer to produce the different available outputs. This step is best executed on a computer cluster (supported clusters are SGE, SLURM, MOAB and LSF. Tests have been successfully made on SGE and SLURM clusters) but will work on a single machine as well (although it will be much slower).

testSequenzaRaw

Generate a quantitative histogram from paired (tumor / normal) bams:

DOCKER_OR_PYTHON -f aCNViewer_DATA/wes/bams/ -t TEST_WES_RAW --refBuild hg19 -w 2000000 -b aCNViewer_DATA/bin --fileType Sequenza --samplePairFile aCNViewer_DATA/wes/bams/sampleFile.txt [--useCustomPloidies USE_CUSTOM_PLOIDIES]

==Here is the full command:==

DOCKER_OR_PYTHON -f BAM_DIR -t OUTPUT_DIR --refBuild REF_BUILD -b BIN_DIR --fileType Sequenza --samplePairFile SAMPLE_PAIR_FILE [-r REF_FILE] [--byChr 1] [-n NB_THREADS] [--createMpileUp CREATE_MPILEUP] [--pattern BAM_FILE_PATTERN] [-M MEMORY] [GENERAL_PLOT_OPTIONS] [HISTOGRAM_OPTIONS] [GISTIC_OPTIONS] [HEATMAP_DENDRO_OPTIONS]
where:

  • BAM_DIR is the folder containing the paired bam files
  • BAM_FILE_PATTERN is an optional parameter which default value is .bam
  • CHR_SIZE_FILE
  • CENTROMERE_FILE
  • WINDOW_SIZE
  • SAMPLE_PAIR_FILE is a tab-delimited file with the following three column names:
    • idvdName
    • sampleName
    • type which should either be T for tumoral samples or N for normal samples
  • REF_FILE is the reference file in fasta format used to generate the bam files. When REF_BUILD is set, REF_FILE is automatically set to the fasta file present in aCNViewer_DATA/genomes/REF_BUILD.
  • BY_CHR is an optional parameter to indicate whether Sequenza should create seqz (Sequenza intermediate file) files by chromosome or not (the default value is 1)
  • NB_THREADS is an optional parameter specifying the number of cores which will be used for each sample pair to create chromosomal seqz files if BY_CHR has been set to 1. If aCNViewer is ran on a supported computer cluster master node, jobs will be submitted to the cluster. Otherwise, multi-threading will be used run Sequenza.
  • CREATE_MPILEUP is an optional parameter telling Sequenza whether to create intermediate mpileup files when generating results. The default value is 1 and it is recommended not to change its value as Sequenza may freeze in some cases when set to 0.
  • MEMORY: optional argument specifying allocated memory in GB to run Sequenza when using a computer cluster. The default value is 8 (GB) and should work for most WES analysis

TestSequenzaCNVs

Generate quantitative stacked histogram from Sequenza results with a window size of 2Mbp:

aCNViewer_DATA.tar.gz is required to run this example.

DOCKER_OR_PYTHON -f aCNViewer_DATA/wes/ -t TEST_WES_SEQUENZA --refBuild hg19 -w 2000000 -b aCNViewer_DATA/bin --fileType Sequenza

==Here is the full command:==

DOCKER_OR_PYTHON -f SEQUENZA_RES_DIR --fileType Sequenza -t TARGET_DIR --refBuild REF_BUILD -b BIN_DIR [GENERAL_PLOT_OPTIONS] [HISTOGRAM_OPTIONS] [GISTIC_OPTIONS] [HEATMAP_DENDRO_OPTIONS]
where:

  • SEQUENZA_RES_DIR is the folder containing Sequenza results (*_segments.txt)

Processing CNV files

At the moment, ASCAT segment file, PennCNV and Sequenza results can be used as an input to aCNViewer. It is possible however to feed aCNViewer with CNV results from any other softwares as explained in the section below.

Both examples below require to download aCNViewer_DATA.tar.gz.

Ascat file generated from Affymetrix SNP arrays

PennCNV

Generate quantitative stacked histogram from PennCNV results (79 samples from Hapmap3):

DOCKER_OR_PYTHON -f aCNViewer_DATA/pennCNV/hapmap3.rawcnv -t TEST_PENN_CNV --refBuild hg18 -b aCNViewer_DATA/bin --lohToPlot none

Sequenza segments

OtherCNVformats

CNV results from any software can be processed by aCNViewer if formatted in the ASCAT segment file format i.e. a tab-delimited file with the following columns:

  • sample
  • chr
  • startpos
  • endpos
  • nMajor
  • nMinor

The result file should be sorted according to the following ordered column names: sample, chr, startpos, endpos and chromosome names in the chr column should not contain the prefix chr so chr1 should appear as 1. All CNVs for one indivual should be non overlapping. If there is only a global CNV value v (and this no allele-specific CNV value), nMajor and nMinor can take any value as long as nMajor + nMinor = v. When plotting the quantitative histogram, add option --lohToPlot none to disable LOH plotting.

OutputFiles

ASCAT

When processing raw SNP array data with aCNViewer, ASCAT is used to calculate CNV profiles. These results are saved into a folder named ASCAT in the user selected target directory with the following files:

  • *.segments.txt: file containing ASCAT predicted CNV segments
  • *.ascatInfo.txt: file containing the following ASCAT values for all the samples: aberrantcellfraction, goodnessOfFit, psi and ploidy
  • *.png: the various ASCAT graphical outputs:
File Description
.ASCATprofile.png genome-wide representation of ASCAT CNVs
.ASPCF.png results of segmentation using Allele-Specific Piecewise Constant Fitting
.rawprofile.png genome-wide representation of raw ASCAT CNVs
.sunrise.png sunrise plot showing the optimal solution of tumor ploidy and percentage of aberrant tumor
.tumour.png representation of LogR and BAF values
tumorSep*.png plot of BAF values
.ascatInfo.txt ASCAT values of aberrantcellfraction, goodnessOfFit, psi and ploidy for all samples
.segments.txt list of all CNVs with the copy number for each allele

GISTIC outputs

For the full list of GISTIC output files, please refer to the section Output Files of the following website. Here are the main output files of interest:

File Description
broad_significance_results.txt The list of broad events with related q-values and frequencies
all_lesions.conf_*.txt the list of all focal events along with their level of significance
amp_* list of all focal amplification events
del_* list of all focal deletion events

Sequenza

The Sequenza results of each sample pair are stored in a folder named TUMOR_NORMAL_sequenza in the sequenza folder and contains the following files:

File Description
*_segments.txt predicted CNVs
*_CP_contours.pdf, *_confints_CP.txt & *_model_fit.pdf inferred cellularity and ploidy
*_alternative_fit.pdf & *_alternative_solutions.txt alternative inferred cellularities and ploidies
*_chromosome_view.pdf chromosome view with mutations, BAF, depth ratio and segments
*_genome_view.pdf genome view of all the CNVs
*_mutations.txt list of detected mutations
*_CN_bars.pdf frequency of all the copy number values

For more information about Sequenza output files, please refer to its user guide.

HistogramOutputs

When generating histograms, 3 text files with the suffix _samples.txt will be created along:

  • one with all the genomic segments
  • one with only the LOH events (file with suffix _loh_samples.txt)
  • one with only the cn-LOH (file with suffix _cnLoh_samples.txt)

Each file is in the same format with the following columns:

  • CNV key: the relative copy number value compared to the tumor ploidy
  • chrName
  • start: the middle of the segment so the real start is start - segmentLength / 2
  • segmentLength: length of the current segment
  • percentage: the percentage of samples with the relative ploidy value in CNV key for the segment (chrName, [start - segmentLength / 2, start + segmentLength / 2])
  • samples: the list of the samples falling in the above category

The following files are created as well:

  • *_10pc_ploidy.txt is a matrix of segments of 10% chromosomal length for all samples. The last column indicates the calculated ploidy which corresponds to the most frequent ploidy

  • *.R are R scripts used to create the various graphical representations. You can modify and re-run these scripts if you want to further customize your graphical outputs and if aCNViewer do not propose the customizations you are looking for.

Dendrograms and heatmaps

2 folders (relCopyNb and rawCopyNb) will be created and will respectively contain graphs generated from relative copy number values and raw copy number values.

Limitations

aCNViewer has a few limitations including the fact that it does not currently account for intra-tumor heterogeneity. Indeed, having a simultaneous view on the copy number landscape along with the clonality status of these events could help better understand the mechanisms of a disease. Another current limitation of aCNViewer is the absence of a function to compare two groups of samples. One simple way to do that, though, would be to generate the quantitative histograms for both groups separately and compare these plots (as we did in Fig 2 of the article below).

Citation

aCNViewer: comprehensive genome-wide visualization of absolute copy number and copy neutral variations. Victor Renault, Jörg Tost, Fabien Pichon, Shu-Fang Wang-Renault, Eric Letouzé, Sandrine Imbeaud, Jessica Zucman-Rossi, Jean-François Deleuze & Alexandre How-Kit. PLoS One. 2017 Dec 19;12(12):e0189334. doi: 10.1371/journal.pone.0189334. eCollection 2017.

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