# Call SNPs and small indels

at least 4 cores/threads available
sequence alignments: .bam coordinate-sorted
- sample name: a-z 0-9 . _ - case insensitive
genome assembly in FASTA format: .fasta .fa .fasta.gz .fa.gz case insensitive
optional sample grouping file

This file is optional and useful if you want variant calling to happen on a per-population level.

takes the format of sample tab group
- spaces can be used as delimeters too
the groups can be numbers or text (i.e. meaningful population names)
you can comment out lines with # for Harpy to ignore them
create with harpy template groupings or manually
if created with harpy template groupings , all the samples will be assigned to group pop1, so make sure to edit the second column to reflect your data correctly.

example file for --populations
sample1 pop1
sample2 pop1
sample3 pop2
sample4 pop1
sample5 pop3
#sample5 pop4

known quirk

There's an unusual error on the Snakemake side of things that happens when the name of a sample and population are identical. It has been unclear how to resolve this issue, so to protect yourself, it's best to make sure the population names are different from the sample names. A simple fix would be to use underscores (_) to differentiate the population name.

After reads have been aligned, e.g., with harpy align , you can use those alignment files (.bam) to call variants in your data. Harpy can call SNPs and small indels using bcftools mpileup or with freebayes. You can call SNPs with the snp module:

usage
harpy snp method OPTIONS... REFERENCE INPUTS...

examples | calling variants with default settings
# call variants with mpileup
harpy snp mpileup --threads 20 genome.fasta Align/bwa

# call variants with freebayes
harpy snp freebayes --threads 20 genome.fasta Align/bwa

# Running Options

In addition to the common runtime options , the snp module is configured using these command-line arguments:

argument	default	description
`REFERENCE` `-g`		required Reference genome used for variant calling
`INPUTS`		required Files or directories containing input BAM files
`--extra-params` `-x`		Additional mpileup/freebayes arguments, in quotes
`--ploidy` `-n`	`2`	Ploidy of samples
`--populations` `-p`		Tab-delimited file of sample<tab>group
`--regions` `-r`	`50000`	Regions to call variants on (see below)

# ploidy

If you are calling haploid or diploid samples, using either mpileup or freebayes will be comparable. However, if you need to call SNPs in polyploids (ploidy >2), then you will need to use freebayes, since mpileup does not call variants for ploidy greater than 2.

# regions

The --regions (-r) option lets you specify the genomic regions you want to call variants on. Keep in mind that mpileup uses 1-based positions for genomic intervals, whereas freebayes uses 0-based positions. Harpy will perform variant calling in parallel over these invervals and they can be specified in three ways:

input: an integer to make fixed-size genomic intervals

example: harpy snp -r 25000 -g genome.fasta data/mapped

This is the default method (-r 50000), where Harpy will create 50 kbp non-overlapping genomic intervals across the entire genome. Intervals towards the end of contigs that are shorter than the specified interval size are still used. These invervals look like:

chromosome_1    1   50000
chromosome_1    50001 100000
chromosome_1    100001  121761    <- reached the end of the contig

input: a single region in the format contig:start-end

example: harpy snp -r chrom1:2000-15000 -g genome.fasta data/mapped

Following the mpileup and freebayes format, you can specify a single genomic interval of interest to call variants on. This interval must be in the format contig:start-end where:

contig is the exact name of a contig in the supplied genome
start is an integer specifying the start position of the interval for that contig
end is an integer specifying the end position of the interval for that contig

input: a tab (or space) delimited file of contigs and positions

example: harpy snp -r data/positions.txt -g genome.fasta data/mapped

A BED-like file of contig<whitespace>start<whitespace>end can be provided to call variants on only specific genomic intervals. This file will look like:

chromosome_1    1   45000
chromosome_1    723123 999919
chromosome_5    22421   564121

# populations

Grouping samples changes the way the variant callers computes certain statistics when calling variants. If you have reason to believe there is a biologically meaningful grouping scheme to your samples, then you should include it.

# SNP calling workflow

The workflow is parallelized over genomic intervals (--regions). All intermediate outputs are removed, leaving you only the raw variants file (in .bcf format), the index of that file, and some stats about it.

# SNP workflows

The mpileup and call modules from bcftools (formerly samtools) or Freebayes are used to call variants from alignments. Both are very popular variant callers to call SNPs and small indels.

graph LR
    subgraph Inputs
        aln[BAM alignments]:::clean---gen[genome]:::clean
    end
    subgraph mpileup
        B([mpileup]):::clean-->C([bcftools call]):::clean
    end
    subgraph freebayes
        z([freebayes]):::clean
    end
    Inputs --> mpileup
    Inputs --> freebayes
    C-->D([index BCFs]):::clean
    D-->E([combine BCFs]):::clean
    z --> D
    C-->E
    E-->F([realign indels]):::clean
    style Inputs fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px,rx:10px,ry:10px
    style mpileup fill:#b0c1b3,stroke:#9eada1,stroke-width:2px,rx:10px,ry:10px
    style freebayes fill:#bcb7cd,stroke:#a9a4b8,stroke-width:2px,rx:10px,ry:10px
    classDef clean fill:#f5f6f9,stroke:#b7c9ef,stroke-width:2px

The default output directory is SNP/mpileup or SNP/freebayes(depending on workflow) with the folder structure below. Below, contig1 and contig2 are generic contig names from an imaginary genome.fasta for demonstration purposes. The resulting folder also includes a workflow directory (not shown) with workflow-relevant runtime files and information.

SNP/method
├── variants.raw.bcf
├── variants.raw.bcf.csi
├── logs
│   ├── contig1.call.log   # mpileup only
│   ├── contig1.METHOD.log
│   ├── contig2.call.log   # mpileup only
│   ├── contig2.METHOD.log
│   ├── sample.groups
│   ├── samples.files
│   └── samples.names
└── reports
    ├── contig1.stats
    ├── contig2.stats
    ├── variants.raw.html
    └── variants.raw.stats

item	description
`variants.raw.bcf`	vcf file produced from variant calling, contains all samples and loci
`variants.normalized.bcf`	variants, but with indels realigned and duplicates removed
`variants.*.bcf.csi`	index file for `variants.*.bcf`
`logs/*.call.log`	what `bcftools call` writes to `stderr`
`logs/*.METHOD.log`	what `bcftools mpileup` or `freebayes` writes to `stderr`
`logs/sample.groups`	if provided, a copy of the file provided to `--populations` with commented lines removed
`logs/samples.files`	list of alignment files used for variant calling
`logs/samples.names`	list of sample names associated with alignment files used for variant calling
`reports/*.stats`	output of `bcftools stats`
`reports/variants.*.html`	report summarizing variants

By default, Harpy runs mpileup with these parameters (excluding inputs and outputs):

bcftools mpileup --region contigname:START-END --annotate AD --output-type b --ploidy ploidy

The mpileup module of samtools has a lot of command line options. Listing them all here would be difficult to read, therefore please refer to the mpileup documentation to explore ways to configure your mpileup run.

By default, Harpy runs freebayes with these parameters (excluding inputs and outputs):

freebayes -f genome.fasta -r contigname:START-END -L bam.list -p ploidy

Freebayes has a lot of command line options. Listing them all here would be difficult to read, therefore please refer to the freebayes documentation to explore ways to configure your freebayes run.

These are the summary reports Harpy generates for this workflow. You may right-click the image and open it in a new tab if you wish to see the example in better detail.

Variant stats

Summarizes information provided by bcftools stats on the called SNPs and indels.