This is a collection of scripts for a range of genomic data processing and analysis.

Below are notes about some useful tools. Not everything is documented yet, but most scripts have some help information if you type python script.py -h

Contents

• Installation and dependencies
• Processing VCF files
• Filtering genotype files prior to further analysis
• Diversity and divergence analyses in sliding windows
• Allele frequencies per site
• Site frequency spectrum
• Distance matrix
• ABBA-BABA statistics in sliding windows
• Trees for sliding windows
• Classify coding sites

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Installation and dependencies

There is no system installation required. Just download this entire repository using the green "Code" button at the top of this page, or with the bash command git clone https://github.com/simonhmartin/genomics_general.git. If you prefer to move scripts to the directory where you run them, bear in mind that many of these scripts require the script genomics.py to be present in the same directory (or on your PYTHONPATH). The easiest is just to leave them in the main directory and specify the full path to the script when running it from elsewhere.

The only dependency is numpy.

Most of the scripts now run in python 3. Some are still written for puthon 2, but those will be updated soon.

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Processing VCF files

Most of my scripts use a processed .vcf format that I call .geno. This looks something like this:

#CHROM      POS      ind1      ind2      ind3
scaffold1   1        A/A       A/G       G|A
scaffold1   1        N/N       T/T       T|C

Missing data is denoted as N, and phased and unphased genotypes are shown conventionally with | and /.

The script parseVCF.py in the VCF_processing directory, will convert vcf to this format. It has various options for filtering based on read depth, genotype quality or any other flag in the FORMAT column of the vcf.

Example command

python VCF_processing/parseVCF.py -i input.vcf.gz --skipIndels --minQual 30 --gtf flag=DP min=5 max=50 -o output.geno.gz

You can read more about this script in the VCF_processing directory.

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Filtering genotype files prior to further analysis

If you vcf file was not already filtered, or you would like to filter further. The script filtergenotypes.py has many options for filtering. Some examples include:

• Number of individuals with non-missing N/N genotypes at a site (--minCalls)
• Number of alleles observed at a site across all individuals (--minAlleles and --maxAlleles)
• Minor allele count (--minVarCount)
• Distance between sites for thinning (--thinDist)

It requires the script genomics.py to be present in the same directory, or in your Python path.

Example command

python filterGenotypes.py --threads 4 -i input.geno.gz -o output.geno.gz --minAlleles 2 --minCalls 10 --thinDist 1000

Notes

python filterGenotypes.py -h will print a full list of command options.

By default, this script assumes that the .geno input file is is encoded as diploid genotypes with a phase operator (/ or |):

scaffold1  1        A/A       G/G       G|A

You can specify a different input formats using the -if, but this is not recommended.

You can also specify various putput formats using -of.

Output format	Description	Example
phased (default)	Alleles separates by a phase operator. This doesn't mean the phase is known, just that it is indicated	A/A G/G G|A
diplo	For diploids only. Genotypes are single bases denoting the diploid genotype, using ambiguity codes for heterozygotes	A G R
alleles	as above but without the phase operator	AA GG GA
randomAllele	Randomly pick one allele per individual	A G A
coded	Coded numerically as in the VCF	0/0 1/1 1|0
bases	Separate the alleles for each individual into different columns and also give different headers for each	A A G G G A
counts	Count of the minor allele in each individual	0 2 1

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Diversity and divergence analyses in sliding windows

The script popgenWindows.py computes some standard population genomic statistics in sliding windows: pi, F_ST and D_XY. It requires the script genomics.py to be present in the same directory, or in your Python path.

Example command

python popgenWindows.py -w 50000 -m 5000 -g input.geno.gz -o output.csv.gz -f phased -T 5 -p popA A1,A2,A3,A4 -p popB B1,B2,B3,B4,B6,B6 -p popC -p popD --popsFile pops.txt 

Notes

python popgenWindows.py -h Will print a full list of command arguments.

• Input is a .geno file as shown above. This can be gzipped (.geno.gz). Output is a .csv. If you add .gz it will be gzipped.

• Genotype encoding is indicated by the -f flag. -f phased is normally used, see the table above. Other options are -f haplo for haploid data (although phased will also interpret haploid data correctly), -f diplo and f pairs which is like the alleles output in the table above, but assumes diplid data.

• There are three options for defining the windows to be analysed, using the --windType argument.

Wndow Type	Description
coordinate	Windows will cover a fixed range in the genome, which is defined as the window size. If there is missing data, this can lead to variable numbers of sites used for each window.
sites	Each window will have the same number of sites. If there is missing data, this can lead to different absolute sizes for windows in terms of genome coordinates.
predefined	This will analyse predefined windows provided using the --windCoords flag.

• You can either include sample names after the population name, separated by commas, or provide only the population name, along with a populations file, with the flag --popsFile , which has two columns: the first gives sample names and teh second gives population name:

C1  popC
C2  popC
C3  popC
C4  popC
D1  popD
D2  popD
D3  popD
D4  popD

• The most common source of errors here involve the -m (--minSites) flag. If you are useing coordinate windows and have any sites with missing data, then -m must be set to a value smaller than the window size. If you have reduced representation data such as RADseq, you will need a much lower -m value (more like 1% of the window size or even less).

• If some samples are haploid and others are diploid, you can use one of the diploid formats, but indicate that certain samples are haploid by listing them after the --haploid flag. The script will force them to have haploid genotyps, and any apparently heterozygous genotype will be converted to N.

• The script can run on multiple cores (-T flag). Try different numbers, as using too many can slow the script down (due to the difficulty in sorting the outputs coming from the different cores).

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Allele frequencies per site

The script freq.py computes allele frequencies at each site, either for each base, the minor allele, or the derived allele (if an outgroup is provided). It requires the script genomics.py to be present in the same directory, or in your Python path.

Example command

python freq.py -g input.geno.gz -p pop1 -p pop2 --popsFile populations.txt --target derived --threads 10

Notes

• Input is a .geno file as shown above. This can be gzipped (.geno.gz). Output is a .csv. If you add .gz it will be gzipped.

• If you do not include the --target option, the script will export the count (i.e. an integer) of each base (A, C, G, T) for ech population.

• If you set --target, you have two options

Wndow Type	Description
--target minor	Export the frequency of the minor (rarer) allele at each site (based on all included individuals). This can be at most 0.5 for the whole dataset, but can exceed 0.5 in certain populations
--target derived	Export the frequency of the derived allele. NOTE: this assumes that the last population listed with -p is the outgroup.

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Site frequency spectrum

The script sfs.py computes site frequency spectrum (SFS, also called the allele frequency spectrum) from input variants. It requires the script genomics.py to be present in the same directory, or in your Python path.

The input for sfs.py is produced by freq.py (see above). These two scripts can be combined with a pipe.

Example commands

#1D folded (derived allele) sfs
python freq.py -g input.geno.gz -f phased --threads 10 -p pop1 -p pop2 -p outgroup | \
python sfs.py --inputType baseCounts -p pop1 -p pop2 -p outgroup \
--FSpops pop1 pop2 --polarized --subsample 10 10 --pref mydata. --suff .subsample10.sfs

#2D unfolded (minor allele) SFS
python freq.py -g input.geno.gz -f phased --threads 10 -p pop1 -p pop2 | \
python sfs.py --inputType baseCounts -p pop1 -p pop2 \
--FSpops pop1 pop2 --doPairs --subsample 10 10 --pref mydata. --suff .subsample10.sfs

Notes

• Input is a .geno file as shown above. This can be gzipped (.geno.gz).

• Output is a table giving the number of sites with each allele count.

• --polarized will produce an unfolded SFS assuming the final population named is the outgroup

• --doPairs, --doTrios and doQuartets will also export the 2D, 3D and 4D SFS for all pairs, trios or quartets of populations.

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Distance matrix

The script distMat.py computes a distance matrix among all pairs of individuals. This can be computed either for the entire input file or in windows, as in the popgenWindows script above. This works for samples of any ploidy or mix of ploidies. For ploidy > 1, the pairwise diatance will be the average diatance among all haplotypes in the two individuals.

Example Command

python distMat.py -g input.geno.gz -f phased --windType cat -o output.dist

python distMat.py -h will print a list of command options.

Notes

• This script shares several command arguments with popgenWindows.py. And input formats are the same. Please see the notes for that script above.

• Output format has three otptions: raw, phylip and nexus.

• To make a single matrix for the entire input file (i.e. not making windows and ignoring scaffold bounaries), use --windType cat

• If using --windType cat, the entire input file will be read into memory. This often leads to RAM issues for large files. One option is to filter your data hard before doing this analysis using filterGenotypes.py.

• To make separate matrices for windows, use one of the window type options described above. There will still be a single output file, but with separate matrices separated by blank lines.

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ABBA-BABA statistics in sliding windows

The script ABBABABAwindows.py performs analyses described in Martin et al. 2015, MBE, compurting the D statistic and f estimators in windows across the genome. Like the script above, it requires genomics.py.

Example command

python ABBABABAwindows.py -g /zoo/disk1/shm45/vcf/set62/set62.chr21.DP5GQ30.AN100MAC1.diplo.gz -f phased -o output.csv -w 100000 -m 100 -s 100000 -P1 A -P2 B -P3 C -O D -T 10 --minData 0.5 --popsFile pops.txt --writeFailedWindows --polarize &

python ABBABABAwindows.py -h Will print a full list of command arguments.

Notes

• This script shares several command arguments with popgenWindows.py. And input formats are the same. Please see the notes for that script above.

• As above, you can either include sample names after the population name, separated by commas, or provide a populations file, which has two columns: the first gives sample names and teh second gives population name.

• f_d gives meaningless values (<0 or >1) if D is negative. If there is no excess of shared derived alleles between P2 and P3 (indicated by a positive D), then the excess cannot be quantified. f_d values for windows with negative D should therefore either be discarded or converted to zero, depending on your hypothesis.

• If you are interested in shared variation between P3 and P2 (positive D) or between P3 and P1 (negative D), then f_d might not be the best approach. f_dM is an alternatve statistic, devised by Milan Malinsky that is better suited to this scenario. It gives positive values for introgression between P3 and P2 and negative values for introgression between P3 and P1. However, my simulation tests show that f_dM tends to underestimate the admixture proportion. Also, note that if introgression occurred between both P3 and P2 and P3 and P1 at the same locus, then it cannot be accurately quantified, because the signal of shared variation depends on either P1 or P2 being uninvolved.

• If a small number of SNPs is used per window, stochastic errors can cause f_d to have meaningless values even when D is positive. Therefore, try to use a window size that allows at least 100 biallelic SNPs per window (see the sitesUsed column to see the number of biallelic SNPs available).

Output

Column Header	Description
scaffold	The scaffold the window is on (all windows are on a single scaffold)
start	window start position (inclusive)
end	window end position (NOTE, this can exceed the length of the scaffold)
sites	Number of genotypes sites in the input file in each window
sitesUsed	number of sites used to compute statistics (biallelic SNPs)
ABBA	Pseudo count of ABBA sites (including polymorphic sites) (See Martin et al. 2015 Equation 2)
BABA	Pseudo count of BABA sites (including polymorphic sites) (See Martin et al. 2015 Equation 3)
D	D statistic (see Durand et al. 2011 Equation 2)
fd	fd admixture estimation (See Martin et al. 2015 Equation 6)
fdM	Malinsky's modified statistic, fdM to accomodate admixture between either P1 and P3 or P2 and P3 (See Malinsky et al. 2015 Supplementart Material Page 8)

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Trees for sliding windows

Two scripts in the phylo/ directory will make trees in sliding windows: phymlWindows.py and raxmlWindows.py. As the names suggest they use Phyml and RAxML, respectively.

Example command

python phyml_sliding_windows.py -T 10 -g input.phased.geno.gz --prefix output.phyml_bionj.w50 -w 50 --windType sites --model GTR 

python phymlWindows.py -h Will print a full list of command arguments.

Notes

• You need to have Phyml (or RAxML) installed on your machine. You can direct the script to the location of the executable. I recommend using an unthreaded version, since each window tree will run very quickly.

• The window can be defined based on genomic coordinates (--windType coord) or the number of sites (--windType sites). Windows will not cross contig/scaffold boundaries.

• Genotypes need to be in either the phased, haplo or diplo formats shown above (diplo format is not recommended, as heterozygous genotypes will be treated as single genotypes with ambiguous bases, which are ignored by Phyml.

• For the raxml script, you could also use diplo format, although I'm not sure whether the ambiguity codes will be used at all by RAxML. It is certainly better to use phased sequences if you can.

• If diploid genotypes are in the phased format, they will be split into haplotypes, and the suffixes '_A' and '_B' will be added to the sample names to distinguish the haplotypes.

• To make neighbour-joining trees, use the Phyml script, and set --optimise n, which tells it not to do any ML optimisation.

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Classify coding sites

Given a genome annotation file, the script codingSiteTypes.py classifies each site within a codon according to its codon position, synonymous/non-synonymopus (if it's a variant) and degeneracy.

Example Command

python codingSiteTypes.py -a annotation.gff3.gz -f gff -r reference.fasta.gz -v variants.vcf.gz --ignoreConflicts | bgzip > output.coding_site_types.tsv.gz

python distMat.py -h will print a list of command options.

Notes

• You will need a annotation file in either .gff3 or .gtf format. Specify the format using -f gff or -f gtf
• The VCF file is optional. If provided, sites can be identified as synonymus or nonsynonymous variants. It also tends to make the classification of degeneracy more conservative.
• All the input files can be gzipped, but they don't have to be.
• The output has five columns: scaffold, position, codon_position, substitution_type and degeneracy.
• substitution_type can be synonymous (syn) or nonsynonymous (non), or simply NA, if the site is not variable in the dataset, or if no VCF file is provided.
• Degeneracy tells how many of the four possible states at this site will result in the same amino acid for the codon.
• It might be useful to make a BED file for sites of a given type. For example, to make a BED file for four-fold degenerate (4D) sites, you can use: gunzip -c output.coding_site_types.tsv.gz | awk 'BEGIN {OFS="\t"}; $5=="4" {print($1,$2-1,$2)}' > output.4Dsites.bed