1. Introduction

ANACONDA is a tool designed for detecting and annotating somatic copy number alterations (CNVs) by integrating four state-of-the-art CNV-calling methods: ExomeCNV, FREEC, ADTEx and EXCAVATOR. In the framework of ANACONDA, configurations of all methods and CNV-calling procedures are automatically done, which is very convenient for non-professional users. Furthermore, the common CNVs detected by multiple algorithms are extracted and further annotated in several dimensions using currently available resources including xxx. Finally, an html file is generated to provide a detailed display of the information about parameter settings, detected CNVs, annotation results and etc.

2. Requirements

√ Unix-like systems
√ R3.0+: The main analysis of CNVs in ExomeCNV, ADTEx and EXCAVATOR is done using R statistical language.(Note: R3.3 doesn’t support some needed packages, we have run ANACONDA successfully by R3.2.2 and R3.2.4. So we highly recommend R3.2.)
√ Jdk8+: Java language is required by ExomeCNV to derive depth of coverage
√ Other necessary tools: gcc and g++ to compile source code

3. Installation

The installation step prepares the running environments for the adopted CNV-calling methods including: 1) check if programing environments are ready, and 2) install dependent packages required by each method. We provide a script named “install” in the top directory of ANACONDA package to complete these steps, users can type following commands to install the software:
chmod +x install
./install
We suggest that users run above two commands as root user, otherwise it may throw an error of “permission denied”. During installation, any errors occurred are recorded in file “log.err”, users can check this file to find more details. After the installation, the main program of ANACONDA is generated in “bin” directory.
Note: Because R package ‘Hmisc’ has so many dependencies, users should install it manually. You can type following command in R shell to install it:
install.packages('Hmisc',dependencies=T,repos="http://cran.rstudio.com/")

4. Configuration

The configuration step setups all required parameters and provides inputs for each algorithm. Generally, the parameters are divided into five groups:
♦ General. The common parameters requited by multiple methods are specified in this part. These parameters including selected methods for calling CNVs, tumor/normal BAM file, the sequence file of reference genome, target capture file, tumor ploidy and etc.
♦ ExomeCNV. The specific parameters for ExomeCNV: (i) parameters for calling exon-level CNVs, and (ii) parameters for combing exons.
♦ FREEC. Parameter settings for FREEC. These parameters will be used to automatically generate the specific configuration file for FREEC
♦ ADTEx. The only one parameter needs to be specified for ADTEx, namely minimum read depth in control sample.
♦ EXCAVATOR. Parameters uniquely required by EXCAVATOR are listed in this part
We provide a detailed description of all parameters as follows:

Group	Parameter	Description	Possible values
General	methods	Methods for CNV calling	Default: ExomeCNV, FREEC, ADTEx, EXCAVATOR Ex: ExomeCNV, ADTEx
	tumor	Tumor BAM file	Ex: /path/to/tumor.bam
	normal	Normal BAM file	Ex: /path/to/normal.bam
	ref	Genome reference sequence file	Ex: /path/to/ref.fa
	refVersion	Version of human genome	Ex: hg18 or hg19
	target	File with capture regions	Ex: /path/to/target.bed
	out	Output directory to save results	Ex: /path/to/outputDir
	ploidy	Tumor ploidy	Default: 2 Ex: 2 or 3 or 4
	purity	Tumor purity	Default: 1.0 Ex: value between 0 and 1
	cnaCalledMethods	A CNV is considered to be a common CNV if it is called by multiple methods	Default: 1 Ex: a value belongs to [1,2,…,#methods]
	geneMinCoverage	Minimum percentage of a gene to be covered by a CNV	Default: 0.7 Ex: value between 0.5 and 1.0
ExomeCNV	min.spec1	Minimum specificity for calling exon-level CNVs	Default: 0.9999 Ex: value between 0 and 1
	min.sens1	Minimum sensitivity for calling exon-level CNVs	Default: 0.9999 Ex: value between 0 and 1
	option1	Optimization Strategy for calling exon-level CNVs	Default: "spec" Ex: "spec" or "auc"
	min.spec2	Minimum sensitivity for combining exons	Default: 0.99 Ex: value between 0 and 1
	min.sens2	Minimum sensitivity for combining exons	Default: 0.99 Ex: value between 0 and 1
	Option2	Optimization Strategy for combining exons	Default: "auc" Ex: "spec" or "auc"
FREEC	BedGraphOutput	Set TRUE if you want an additional output in BedGraph format for the UCSC genome browser	Default: FALSE
	breakPointThreshold	Positive value of threshold for segmentation of normalized profiles	Default: 0.8
	breakPointType	Desired behavior in the ambiguous regions	Default: 2
	chrFiles	Path to the directory with chromosomes fasta files	Ex: /path/to/hg18
	contaminationAdjustment	Set TRUE to correct for contamination by normal cells	Default: FALSE
	forceGCcontentNormalization	Simply model "sample RC ~ Control RC"	Fixed value: 0
	intercept	Intercept of polynomial	Fixed value: 0
	minCNVlength	Minimal number of consecutive windows to call a CNV	Default: 3
	maxThreads	Number of threads	Default: 1
	degree	Degree of polynomial	Fixed value: 1
	readCountThreshold	Threshold on the minimal number of reads per window in the control sample	Default: 10
	sex	Sample sex	"sex=XX" will exclude chr Y from the analysis "sex=XY" will not annotate one copy of chr X and Y as a loss
	mateOrientation	Format of reads	Fixed value: 0
ADTEx	minReadDepth	Threshold on the minimum read depth in the control sample	Default: 10
EXCAVATOR	wigFile	Mappability file	Ex: /path/to/hg18_uniqueome.Wig
	mapQ	Mapping quality for BAM file filtering	Default: 0
	Omega	Omega parameter for the HSLM algorithm	Default: 0.1
	Theta	Theta parameter for the HSLM algorithm	Default: 1e-4
	D_norm	D_norm parameter for the HSLM algorithm	Default: 10e6
	Thre_d	Threshold d for the truncated gaussian distribution of the FastCall Calling algorithm	Default: 0.5
	Thre_u	Threshold u for the truncated gaussian distribution of the FastCall Calling algorithm	Default: 0.35
	minSeglen	Segment with a number of exons smaller than a threshold are filtered out	Default: 4

5. Running ANACONDA

Running the main program of ANACONDA only needs one argument that is the configuration file. Type following command to run ANACONDA:
./bin/ANACONDA /path/to/configfile
For example:
./bin/ANACONDA ./config/config.txt