Getting started


  1. Create a sample configuration file for your project (substitute the example BAM and fastq names below with the full path to your sample files): -w template gatk-variant project1 sample1.bam sample2_1.fq sample2_2.fq

    This uses a standard template (GATK best practice variant calling) to automate creation of a full configuration for all samples. See Automated sample configuration for more details on running the script, and manually edit the base template or final output file to incorporate project specific configuration. The example pipelines provide a good starting point and the Sample information documentation has full details on available options.

  2. Run analysis, distributed across 8 local cores: bcbio_sample.yaml -n 8
  3. Read the Configuration documentation for full details on adjusting both the sample and system configuration files to match your experiment and computational setup.

Project directory

bcbio encourages a project structure like:

├── config
├── final
└── work

with the input configuration in the config directory, the outputs of the pipeline in the final directory, and the actual processing done in the work directory. Run the script from inside the work directory to keep all intermediates there. The final directory, relative to the parent directory of the work directory, is the default location specified in the example configuration files and gets created during processing. The final directory has all of the finished outputs and you can remove the work intermediates to cleanup disk space after confirming the results. All of these locations are configurable and this project structure is only a recommendation.


There are 3 logging files in the log directory within your working folder:

  • bcbio-nextgen.log High level logging information about the analysis. This provides an overview of major processing steps and useful checkpoints for assessing run times.
  • bcbio-nextgen-debug.log Detailed information about processes including stdout/stderr from third party software and error traces for failures. Look here to identify the status of running pipelines or to debug errors. It labels each line with the hostname of the machine it ran on to ease debugging in distributed cluster environments.
  • bcbio-nextgen-commands.log Full command lines for all third party software tools run.

Example pipelines

We supply example input configuration files for validation and to help in understanding the pipeline.

Whole genome trio (50x)

This input configuration runs whole genome variant calling using bwa, GATK HaplotypeCaller and FreeBayes. It uses a father/mother/child trio from the CEPH NA12878 family: NA12891, NA12892, NA12878. Illumina’s Platinum genomes project has 50X whole genome sequencing of the three members. The analysis compares results against a reference NA12878 callset from NIST’s Genome in a Bottle initiative.

To run the analysis do:

mkdir -p NA12878-trio-eval/config NA12878-trio-eval/input NA12878-trio-eval/work
cd NA12878-trio-eval/config
cd ../input
cd ../work ../config/NA12878-trio-wgs-validate.yaml -n 16

This is a large whole genome analysis and meant to test both pipeline scaling and validation across the entire genome. It can take multiple days to run depending on available cores. It requires 300Gb for the input files and 1.3Tb for the work directory. Smaller examples below exercise the pipeline with less disk and computational requirements.

We also have a more extensive evaluation that includes 2 additional variant callers, Platypus and samtools, and 3 different methods of calling variants: single sample, pooled, and incremental joint calling. This uses the same input data as above but a different input configuration file:

mkdir -p NA12878-trio-eval/work_joint
cd NA12878-trio-eval/config
cd ../work_joint ../config/NA12878-trio-wgs-joint.yaml -n 16

Exome with validation against reference materials

This example calls variants on NA12878 exomes from EdgeBio’s clinical sequencing pipeline, and compares them against reference materials from NIST’s Genome in a Bottle initiative. This supplies a full regression pipeline to ensure consistency of calling between releases and updates of third party software. The pipeline performs alignment with bwa mem and variant calling with FreeBayes, GATK UnifiedGenotyper and GATK HaplotypeCaller. Finally it integrates all 3 variant calling approaches into a combined ensemble callset.

This is a large full exome example with multiple variant callers, so can take more than 24 hours on machines using multiple cores.

First get the input configuration file, fastq reads, reference materials and analysis regions:

mkdir -p NA12878-exome-eval
cd NA12878-exome-eval

Finally run the analysis, distributed on 8 local cores, with:

cd work ../config/NA12878-exome-methodcmp.yaml -n 8

The grading-summary.csv contains detailed comparisons of the results to the NIST reference materials, enabling rapid comparisons of methods.

Cancer tumor normal

This example calls variants using multiple approaches in a paired tumor/normal cancer sample from the ICGC-TCGA DREAM challenge. It uses synthetic dataset 3 which has multiple subclones, enabling detection of lower frequency variants. Since the dataset is freely available and has a truth set, this allows us to do a full evaluation of variant callers.

To get the data:

mkdir -p cancer-dream-syn3/config cancer-dream-syn3/input cancer-dream-syn3/work
cd cancer-dream-syn3/config
cd ../input

Run with:

cd ../work ../config/cancer-dream-syn3.yaml -n 8

The configuration and data file has downloads for exome only and whole genome analyses. It enables exome by default, but you can use the larger whole genome evaluation by uncommenting the relevant parts of the configuration and retrieval script.

Cancer-like mixture with Genome in a Bottle samples

This example simulates somatic cancer calling using a mixture of two Genome in a Bottle samples, NA12878 as the “tumor” mixed with NA24385 as the background. The Hartwig Medical Foundation and Utrecht Medical Center generated this “tumor/normal” pair by physical mixing of samples prior to sequencing. The GiaB FTP directory has more details on the design and truth sets. The sample has variants at 15% and 30%, providing the ability to look at lower frequency mutations.

To get the data:


Then run the analysis with:

cd work ../config/cancer-giab-na12878-na24385.yaml -n 16

Structural variant calling – whole genome NA12878 (50x)

This example runs structural variant calling with multiple callers (Lumpy, Manta and CNVkit), providing a combined output summary file and validation metrics against NA12878 deletions. It uses the same NA12878 input as the whole genome trio example.

To run the analysis do:

mkdir -p NA12878-sv-eval
cd NA12878-sv-eval
cd work ../config/NA12878-sv.yaml -n 16

This is large whole genome analysis and the timing and disk space requirements for the NA12878 trio analysis above apply here as well.

RNAseq example

This example aligns and creates count files for use with downstream analyses using a subset of the SEQC data from the FDA’s Sequencing Quality Control project.

Get the setup script and run it, this will download six samples from the SEQC project, three from the HBRR panel and three from the UHRR panel. This will require about 100GB of disk space for these input files. It will also set up a configuration file for the run, using the templating system:


Now go into the work directory and run the analysis:

cd seqc/work ../config/seqc.yaml -n 8

This will run a full scale RNAseq experiment using Tophat2 as the aligner and will take a long time to finish on a single machine. At the end it will output counts, Cufflinks quantitation and a set of QC results about each lane. If you have a cluster you can parallelize it to speed it up considerably.

A nice looking standalone report of the bcbio-nextgen run can be generated using bcbio.rnaseq. Check that repository for details.

Human genome build 38

Validate variant calling on human genome build 38, using two different builds (with and without alternative alleles) and three different validation datasets (Genome in a Bottle prepared with two methods and Illumina platinum genomes). To run:

mkdir -p NA12878-hg38-val
cd NA12878-hg38-val
cd work ../config/NA12878-hg38-validate.yaml -n 16

Whole genome (10x)

An input configuration for running whole gnome variant calling with bwa and GATK, using Illumina’s Platinum genomes project (NA12878-illumina.yaml). See this blog post on whole genome scaling for expected run times and more information about the pipeline. To run the analysis:

  • Create an input directory structure like:

    ├── config
    │   └── NA12878-illumina.yaml
    ├── input
    └── work
  • Retrieve inputs and comparison calls:

    cd input
  • Retrieve configuration input file:

    cd config
  • Run analysis on 16 core machine:

    cd work ../config/NA12878-illumina.yaml -n 16
  • Examine summary of concordance and discordance to comparison calls from the grading-summary.csv file in the work directory.

Test suite

The test suite exercises the scripts driving the analysis, so are a good starting point to ensure correct installation. Tests use the pytest framework. The tests are available in the bcbio source code:

$ git clone

There is a small wrapper script that finds the py.test and other dependencies pre-installed with bcbio you can use to run tests:

$ cd tests
$ ./

You can use this to run specific test targets:

$ ./ cancer
$ ./ rnaseq
$ ./ devel
$ ./ docker

Optionally, you can run pytest directly from the bcbio install to tweak more options. It will be in /path/to/bcbiio/anaconda/bin/py.test. Pass -s to py.test to see the stdout log, and -v to make py.test optput more verbose. The tests are marked with labels which you can use to run a specific subsets of the tests using the -m argument:

$ py.test -m rnaseq

To run unit tests:

$ py.test tests/unit

To run integration pipeline tests:

$ py.test tests/integration

To run tests which use bcbio_vm:

$ py.test tests/bcbio_vm

To see the test coverage, add the --cov=bcbio argument to py.test.

By default the test suite will use your installed system configuration for running tests, substituting the test genome information instead of using full genomes. If you need a specific testing environment, copy tests/data/automated/post_process-sample.yaml to tests/data/automated/post_process.yaml to provide a test-only configuration.