# Quick start¶

If you would like to quickly try CNVkit without installing it, try our app on DNAnexus.

## Install CNVkit¶

https://github.com/etal/cnvkit

1. Your species’ reference genome sequence, in FASTA format [required]
2. Gene annotation database, via RefSeq or Ensembl, in “flat” format (e.g. refFlat.txt) [optional]

You probably already have the reference genome sequence. If your species’ genome is not available from UCSC, use whatever reference sequence you have. CNVkit only requires that your reference genome sequence be in FASTA format. Both the reference genome sequence and the annotation database must be single, uncompressed files.

Sequencing-accessible regions: If your reference genome is the UCSC human genome hg19, a BED file of the sequencing-accessible regions is included in the CNVkit distribution as data/access-5kb-mappable.hg19.bed. If you’re not using hg19, consider building the “access” file yourself from your reference genome sequence (say, mm10.fasta) using the access command:

cnvkit.py access mm10.fasta -s 10000 -o access-10kb.mm10.bed


We’ll use this file in the next step to ensure off-target bins (“antitargets”) are allocated only in chromosomal regions that can be mapped.

Gene annotations: The gene annotations file (refFlat.txt) is useful to apply gene names to your baits BED file, if the BED file does not already have short, informative names for each bait interval. This file can be used in the next step.

chr1        1508981 1509154
chr1        2407978 2408183
chr1        2409866 2410095


Then you want refFlat.txt.

Otherwise, if they look like:

chr1        1508981 1509154 SSU72
chr1        2407978 2408183 PLCH2
chr1        2409866 2410095 PLCH2


Then you don’t need refFlat.txt.

## Map sequencing reads to the reference genome¶

If you haven’t done so already, use a sequence mapping/alignment program such as BWA to map your sequencing reads to the reference genome sequence.

You should now have one or BAM files corresponding to individual samples.

## Build a reference from normal samples and infer tumor copy ratios¶

Here we’ll assume the BAM files are a collection of “tumor” and “normal” samples, although germline disease samples can be used equally well in place of tumor samples.

CNVkit uses the bait BED file (provided by the vendor of your capture kit), reference genome sequence, and sequencing-accessible regions along with your BAM files to:

1. Create a pooled reference of per-bin copy number estimates from several normal samples; then
2. Use this reference in processing all tumor samples that were sequenced with the same platform and library prep.

All of these steps are automated with the batch command. Assuming normal samples share the suffix “Normal.bam” and tumor samples “Tumor.bam”, a complete command could be:

cnvkit.py batch *Tumor.bam --normal *Normal.bam \
--targets my_baits.bed --fasta hg19.fasta \
--access data/access-5kb-mappable.hg19.bed \
--output-reference my_reference.cnn --output-dir example/


See the built-in help message to see what these options do, and for additional options:

cnvkit.py batch -h


If you have no normal samples to use for the reference, you can create a “flat” reference which assumes equal coverage in all bins by using the --normal/-n flag without specifying any additional BAM files:

cnvkit.py batch *Tumor.bam -n -t my_baits.bed -f hg19.fasta \
--access data/access-5kb-mappable.hg19.bed \
--output-reference my_flat_reference.cnn -d example2/


In either case, you should run this command with the reference genome sequence FASTA file to extract GC and RepeatMasker information for bias corrections, which enables CNVkit to improve the copy ratio estimates even without a paired normal sample.

If your targets are missing gene names, you can add them here with the --annotate argument:

cnvkit.py batch *Tumor.bam -n *Normal.bam -t my_baits.bed -f hg19.fasta \
--annotate refFlat.txt --access data/access-5kb-mappable.hg19.bed \
--output-reference my_flat_reference.cnn -d example3/


Note

Which BED file should I use?

• target vs. bait BED files: For hybrid capture, the targeted regions (or “primary targets”) are the genomic regions your capture kit attempts to ensure are well covered, e.g. exons of genes of interest. The baited regions (or “capture targets”) are the genomic regions your kit actually captures, usually including about 50bp flanking either side of each target. Give CNVkit the bait/capture BED file, not the primary targets.
• For Whole-Genome Sequencing (WGS), use the batch --method wgs option and optionally give the genome’s “access” file – if not given, it will be calculated from the genome sequence FASTA file.
• For Targeted Amplicon Sequencing (TAS), use the batch --method amplicon option and give the target BED file.

## Next steps¶

You can reuse the reference file you’ve previously constructed to extract copy number information from additional tumor sample BAM files, without repeating the steps above. Assuming the new tumor samples share the suffix “Tumor.bam” (and let’s also spread the workload across all available CPUs with the -p option, and generate some figures):

cnvkit.py batch *Tumor.bam -r my_reference.cnn -p 0 --scatter --diagram -d example4/


The coordinates of the target and antitarget bins, the gene names for the targets, and the GC and RepeatMasker information for bias corrections are automatically extracted from the reference .cnn file you’ve built.

Now, starting a project from scratch, you could follow any of these approaches:

• Run batch as above with all tumor/test and normal/control samples specified as they are, and hope for the best. (This should usually work fine.)

• For the careful: Run batch with just the normal samples specified as normal, yielding coverage .cnn files and a pooled reference. Inspect the coverages of all samples with the metrics command, eliminating any poor-quality samples and choosing a larger or smaller antitarget bin size if necessary. Build an updated pooled reference using batch or coverage and reference (see Copy number calling pipeline), coordinating your work in a Makefile, Rakefile, or similar build tool.

• For the power user: Run batch with all samples specified as tumor samples, using -n by itself to build a flat reference, yielding coverages, copy ratios, segments and optionally plots for all samples, both tumor and normal. Inspect the “rough draft” outputs and determine an appropriate strategy to build and use a pooled reference to re-analyze the samples – ideally coordinated with a build tool as above.

• Use a framework like bcbio-nextgen to coordinate the complete sequencing data analysis pipeline.

See the command-line usage pages for additional visualization, reporting and import/export commands in CNVkit.