Dcon: Estimate DNA contamination

Next-generation sequencing (NGS) of DNA is routinely used in clinical testing. However, the complicated, multistep DNA extraction protocol and the unknown pre-analytic specimen handling often lead to sample-to-sample DNA contaminations. DNA contamination poses substantial challenges in NGS assays and affects diagnosis and treatment if contaminant DNA harbors clinically significant variants.

Dcon is a program to detect and quantify sample-to-sample germline DNA contaminations using the skewed (or shifted) distribution of variant allele frequency (VAF, VAF refers to the fraction of sequencing reads overlapping a genomic coordinate of a DNA variant such as SNP).

Unlike other tools, Dcon does NOT require any prior knowledge such as "genotype" and/or "allele frequencies in human population" calculated from 1000 Genome project. Dcon works on single BAM file and does not require tumor-normal pairs. Therefore, Dcon is also able to detect contamination from DNA sequencing data generated from non-human samples, where allele frequencies and genotype are generally not available.

Getting Started

How Dcon works

Prerequisites

Dcon requires python2.7 and these python packages (Python packages below will be automatically installed, please see instructions below).

Installing

Following instructions here to install pip. Please note: pip is already installed if you're using Python 2 >=2.7.9 or Python 3 >=3.4 binaries downloaded from python.org, but you'll need to upgrade pip.

Users have two ways to install Dcon:

Install Dcon from pypi:
- Open a terminal and type command: pip install Dcon
Install Dcon from local directory
- Download Dcon directly from: this link
- If you download dcon-0.1.8.tar.gz. Go to the directory where dcon-0.1.8.tar.gz was saved. Open a terminal and type command:tar -zxf dcon-0.1.8.tar.gz
- If you download dcon-0.1.8.zip. Go to the directory where dcon-0.1.8.zip was saved. Open a terminal and type command:unzip dcon-0.1.8.zip
- cd dcon-0.1.8
- python setup.py install # Use python2.7

General usage

User provides a BAM file and a VCF file. Variants in VCF file can be called from this particular BAM file or extracted from dbSNP (i.e. extract all SNPs that falling into captured genomic regions, Dcon automatically checks the VAF for each SNP and skips irrelevant SNPs).

python2.7 Dcon.py -b input.bam -v input.vcf -o output

Tips: How do I get a sub-section of a VCF file?

Download and install tabix
Using tabix to get a sub-section of a VCF file.

tabix -h ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20100804/ALL.2of4intersection.20100804.genotypes.vcf.gz 2:39967768-39967768 > output.vcf

General usage instruction is listed below:

Usage: Dcon.py [options]

Dcon: Estimate DNA Contamination from BAM file.



Options:
  --version             show program's version number and exit
  -h, --help            show this help message and exit
  -b BAM_FILE, --bam=BAM_FILE
                        Alignment file in BAM format. BAM file must be indexed
                        and sorted using samTools
                        (http://samtools.sourceforge.net/).
  -v VCF_FILE, --variant=VCF_FILE
                        VCF format file containing *candiate* SNPs (Note: Not
                        every SNP in this file has to be the real SNP in your
                        BAM file. They could be SNPs extracted from
                        dbSNP/1000Genome database, as long as they are located
                        in the captured region. However, too many spurious
                        SNPs will reduce the accuracy.). VCF file can be a
                        plain text, compressed (.gz, .z, .Z, .bz, .bz2 and
                        .gzip2) or remote file (http://, https://, ftp://).
                        Exclusive with '-r'.
  -o OUTPUT_FILE, --output=OUTPUT_FILE
                        Prefix of output files. Will generate two intermediate
                        files: "prefix.SNP.tsv", "prefix.PI.filtered.tsv"
  -m MIN_MAPQ, --mapq=MIN_MAPQ
                        Minimum mapping quality
                        (http://maq.sourceforge.net/qual.shtml). Mapping
                        quality is Phred-scaled probability of an alignment
                        being wrong. default=30
  -q MIN_SEQQ, --seqq=MIN_SEQQ
                        Minimum base phred quality
                        (http://maq.sourceforge.net/qual.shtml). Base quality
                        is Phread-scaled probability of a base calling being
                        wrong. default=30
  -c MIN_COVERAGE, --cvg=MIN_COVERAGE
                        Minimum number of reads supporting variant. default=30
  -n PROCESSOR_NUM, --processor=PROCESSOR_NUM
                        Number of processes. default=1
  -p PROBABILITY_CUT, --prob=PROBABILITY_CUT
                        Cutoff of probability.  Probability is calcualted from
                        Bayesian Gaussian Mixture model to decicde if a SNP is
                        homozygous or heterozygous. default=0.5
  -z ZSCORE_CUT, --zscore=ZSCORE_CUT
                        Cutoff of Z-score. The modified Z-score is calculated
                        from median absolute deviation. SNPs with Z-score
                        greater than this cutoff will be considered as outlier
                        and not used to estimate overall contamination level.
                        default=2.5

Create testing datasets

Targeted-capture sequencing data of 10 chronic myelomonocytic leukemia patients were downloaded from NCBI Sequence Read Archive (SRA).

A total of 45 synthetic datasets were created by mixing the above 10 original datasets in pairwise at 5%, 10%, ..., 45%, 50%. Mixed reads were then aligned to human genome (hg19) using BWA. Below are mixed BAM files from "SRR6756025" and "SRR6756028":

File Name	Mix percentage	Reads from SRR6756025	Reads from SRR6756028	Total Reads	Download Links	MD5Sum of BAM file
SRR6756025_SRR6756028_P05.bam	5%	1,900,360	100,122	2,000,482	bam, bai	0fa86559117ee9f3070986593ff3b667
SRR6756025_SRR6756028_P10.bam	10%	1,800,190	199,734	1,999,924	bam, bai	3e8705858900060836eccf984e4fe778
SRR6756025_SRR6756028_P15.bam	15%	1,700,516	298,978	1,999,494	bam, bai	9ea836d52e7b78ec2e21d7fe8a547bdf
SRR6756025_SRR6756028_P20.bam	20%	1,600,992	399,158	2,000,150	bam, bai	f2ed2b1cfbf89b6331f2a86065248767
SRR6756025_SRR6756028_P25.bam	25%	1,501,164	499,074	2,000,238	bam, bai	3f838b28fa8898d12c6313de82473694
SRR6756025_SRR6756028_P30.bam	30%	1,401,194	600,108	2,001,302	bam, bai	2b9392ef983e1e789b5d818c9f604b19
SRR6756025_SRR6756028_P35.bam	35%	1,300,672	700,448	2,001,120	bam, bai	1d3a0fe4990bfa4da599df44d8f4c25a
SRR6756025_SRR6756028_P40.bam	40%	1,200,038	799,920	1,999,958	bam, bai	e52bbeec296c2f933019a49e59639d46
SRR6756025_SRR6756028_P45.bam	45%	1,099,964	899,424	1,999,388	bam, bai	72c8e3fca913735d1270875a3be59b17
SRR6756025_SRR6756028_P50.bam	50%	999,998	998,942	1,998,940	bam, bai	75562d5c2ae4db37d2c97fe09c368222

Running test

Download BAM files, BAM index files from the table above.
Download the VCF file from here.
Running Dcon to estimate contamination level.


#####################################
# Run Dcon on BAM file mixed at 5%  #
#####################################

$ Dcon.py -v gene83exon.snp.vcf.gz -n 4 -b SRR6756025_SRR6756028_P05.bam -o P05

@ 2018-09-07 13:20:55: Running Dcon v0.1.8
@ 2018-09-07 13:20:55: Read SNPs ...
@ 2018-09-07 13:20:55: Estimating contamination for each variant. Saved to "P05.PI.tsv"
@ 2018-09-07 13:20:55: Building Bayesian Gaussian Mixture model (BGMM) ...
	Converge status: True
	Iterations: 3


@ 2018-09-07 13:20:55: Summerzie BGMM model ...
	Means of homozygous component: 0.040164
	Means of heterozygous component: 0.745609
	Weight of homozygous component: 0.431413
	Weight of heterozygous component: 0.568587


@ 2018-09-07 13:20:55: Classify variants ...

@ 2018-09-07 13:20:55: Writing to P05.PI.tsv ...
@ 2018-09-07 13:20:55: Filter outlier SNPs ...
@ 2018-09-07 13:20:55: Estimating overall contamination using maximum likelihood estimation (MLE) ...
@ 2018-09-07 13:20:55: Estimating contamination from homozygous SNPs ...


Overall contamination level of SRR6756025_SRR6756028_P05 is 0.051


#####################################
# Run Dcon on BAM file mixed at 10% #
#####################################

$ con.py -v gene83exon.snp.vcf.gz -n 4 -b SRR6756025_SRR6756028_P10.bam -o P10

@ 2018-09-07 13:23:08: Running Dcon v0.1.8
@ 2018-09-07 13:23:08: Read SNPs ...
@ 2018-09-07 13:23:08: Estimating contamination for each variant. Saved to "P10.PI.tsv"
@ 2018-09-07 13:23:08: Building Bayesian Gaussian Mixture model (BGMM) ...
	Converge status: True
	Iterations: 8


@ 2018-09-07 13:23:08: Summerzie BGMM model ...
	Means of homozygous component: 0.067398
	Means of heterozygous component: 0.702934
	Weight of homozygous component: 0.421991
	Weight of heterozygous component: 0.578009


@ 2018-09-07 13:23:08: Classify variants ...

@ 2018-09-07 13:23:08: Writing to P10.PI.tsv ...
@ 2018-09-07 13:23:08: Filter outlier SNPs ...
@ 2018-09-07 13:23:08: Estimating overall contamination using maximum likelihood estimation (MLE) ...
@ 2018-09-07 13:23:08: Estimating contamination from homozygous SNPs ...


Overall contamination level of SRR6756025_SRR6756028_P10 is 0.094


#####################################
# Run Dcon on BAM file mixed at 15% #
#####################################

$ Dcon.py -v gene83exon.snp.vcf.gz -n 4 -b SRR6756025_SRR6756028_P15.bam -o P15

@ 2018-09-07 13:23:51: Running Dcon v0.1.8
@ 2018-09-07 13:23:51: Read SNPs ...
@ 2018-09-07 13:23:51: Estimating contamination for each variant. Saved to "P15.PI.tsv"
@ 2018-09-07 13:23:51: Building Bayesian Gaussian Mixture model (BGMM) ...
	Converge status: True
	Iterations: 19


@ 2018-09-07 13:23:51: Summerzie BGMM model ...
	Means of homozygous component: 0.107931
	Means of heterozygous component: 0.687673
	Weight of homozygous component: 0.420852
	Weight of heterozygous component: 0.579148


@ 2018-09-07 13:23:51: Classify variants ...

@ 2018-09-07 13:23:51: Writing to P15.PI.tsv ...
@ 2018-09-07 13:23:51: Filter outlier SNPs ...
@ 2018-09-07 13:23:51: Estimating overall contamination using maximum likelihood estimation (MLE) ...
@ 2018-09-07 13:23:51: Estimating contamination from homozygous SNPs ...


Overall contamination level of SRR6756025_SRR6756028_P15 is 0.145


#####################################
# Run Dcon on BAM file mixed at 20% #
#####################################

$ Dcon.py -v gene83exon.snp.vcf.gz -n 4 -b SRR6756025_SRR6756028_P20.bam -o P20

@ 2018-09-07 13:24:17: Running Dcon v0.1.8
@ 2018-09-07 13:24:17: Read SNPs ...
@ 2018-09-07 13:24:17: Estimating contamination for each variant. Saved to "P20.PI.tsv"
@ 2018-09-07 13:24:17: Building Bayesian Gaussian Mixture model (BGMM) ...
	Converge status: True
	Iterations: 21


@ 2018-09-07 13:24:17: Summerzie BGMM model ...
	Means of homozygous component: 0.137115
	Means of heterozygous component: 0.657750
	Weight of homozygous component: 0.417032
	Weight of heterozygous component: 0.582968


@ 2018-09-07 13:24:17: Classify variants ...

@ 2018-09-07 13:24:17: Writing to P20.PI.tsv ...
@ 2018-09-07 13:24:17: Filter outlier SNPs ...
@ 2018-09-07 13:24:17: Estimating overall contamination using maximum likelihood estimation (MLE) ...
@ 2018-09-07 13:24:17: Estimating contamination from homozygous SNPs ...


Overall contamination level of SRR6756025_SRR6756028_P20 is 0.198

Output files

Dcon will generate 2 intermediate files

prefix.SNP.tsv

column-1: Chromosome ID
column-2: SNP Position
column-3: Allele 1
column-4: Number of reads supporting Allele 1
column-5: Allele 2
column-6: Number of reads supporting Allele 1

prefix.PI.filtered.tsv

column-1: Chromosome ID
column-2: SNP Position
column-3: Allele 1
column-4: Number of reads supporting Allele 1
column-5: Allele 2
column-6: Number of reads supporting Allele 2
column-7: Ratio. {Number of reads supporting less frequent allele}/{Total reads covering this SNP}
column-8: Probability of homozygous SNP
column-9: Probability of heterozygous SNP
column-10: Label. "Hom" or "Het"
column-11: Distance used to rank SNP. Contamination calculated from SNPs with smaller distance is more reliable.
column-12: Contamination percentage calculated from this individual SNP.
column-13: Indicator of whether this SNP has passed outlier filtering.

Example of prefix.SNP.tsv

$ head P05.SNP.tsv

1	36937059	A	126	G	117
1	36937065	A	127	G	124
2	25469502	T	116	C	95
3	105439026	G	477	A	11
3	128199380	G	220	A	14
3	128199662	G	170	A	11
3	128204951	C	97	T	90
3	128205860	C	30	G	15
3	128206618	C	36	A	7
3	136056033	G	671	A	5
...

Example of prefix.PI.filtered.tsv

$ head P05.PI.tsv

Chrom	Ref_pos	Allele_1	Allele_1_count	Allele_2	Allele_2_count	Ratio	Prob_of_Het	Prob_of_Hom	Label	Distance	Contamination_p	Outlier
3	105439026	G	477	A	11	0.0230607966457	0.000182230455487	0.999817769545	Hom	0.0171029321787	0.0450819672131	Pass
3	128199380	G	220	A	14	0.0636363636364	0.000446156678585	0.999553843321	Hom	0.023472634812	0.119658119658	Pass
3	128199662	G	170	A	11	0.0647058823529	0.000458638756417	0.999541361244	Hom	0.0245421535286	0.121546961326	Pass
3	128206618	C	36	A	7	0.194444444444	0.0565034056397	0.94349659436	Hom	0.15428071562	0.325581395349	Fail
3	136056184	A	638	G	11	0.0172413793103	0.000164195828332	0.999835804172	Hom	0.022922349514	0.0338983050847	Pass
3	136088038	G	549	A	9	0.016393442623	0.000161803440411	0.99983819656	Hom	0.0237702862014	0.0322580645161	Pass
3	168801495	C	576	A	12	0.0208333333333	0.000174978971172	0.999825021029	Hom	0.019330395491	0.0408163265306	Pass
3	168801916	C	349	T	4	0.0114613180516	0.000148943307201	0.999851056693	Hom	0.0287024107728	0.0226628895184	Pass
3	168802737	G	278	A	3	0.0107913669065	0.000147326774526	0.999852673225	Hom	0.0293723619179	0.0213523131673	Pass

speed benchmark

CPU model

Intel(R) Core(TM) i7-3720QM CPU @ 2.60GHz

Speed:

When VCF (9265 candidate SNPs within captured regions) file was provided , Dcon took 6 minutes and 5 seconds to estimate DNA contamination from a 177 Mb BAM file containing 2 million alignments. Using multiple threads (-p) will increase speed significantly.

Performance

In below figure, each boxplot is generated from 100 BAM files (Capture sequencing target 37 genes). And each BAM file is purposely mixed from two different samples at pre-defined percentages (10%, 20%, 30%, 40%, 50%). For example, in the leftmost boxplot(green), 10% of reads were from one sample, and 90% of reads were from another sample.

Contributing Authors

Liguo Wang Wang.Liguo@mayo.edu
Tao Ma Ma.Tao@mayo.edu
Rohan Gnanaolivu gnanaolivu.rohandavid@mayo.edu
Jagadheshwar Balan Balan.Jagadheshwar@mayo.edu
Eric Klee Klee Klee.Eric@mayo.edu
Jean-Pierre Kocher kocher.jeanpierre@mayo.edu

License

This project is licensed under the GPL License - see the LICENSE file for details

Acknowledgments

This project is partially supported by the Clinical Genome Sequencing Laboratory (CGSL), Mayo Clinic.

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build		build
dist		dist
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lib		lib
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LICENSE		LICENSE
MANIFEST.in		MANIFEST.in
PKG-INFO		PKG-INFO
README.md		README.md
distribute_setup.py		distribute_setup.py
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setup.py		setup.py

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Dcon: Estimate DNA contamination

Getting Started

How Dcon works

Prerequisites

Installing

General usage

Create testing datasets

Running test

Output files

speed benchmark

Performance

Contributing Authors

License

Acknowledgments

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Dcon: Estimate DNA contamination

Getting Started

How Dcon works

Prerequisites

Installing

General usage

Create testing datasets

Running test

Output files

speed benchmark

Performance

Contributing Authors

License

Acknowledgments

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