VCF overview

VCF overview

Compiled by Julia M.I. Barth, 10 January 2025

Background and Aim
In this exercise, we will explore how the information is stored in a VCF file, and some simple ways of how we can inspect it. This will also serve as a way to practice basic UNIX skills.

The data set

We will work with a data set containing genomic data of 204 individuals of Atlantic cod (Gadus morhua) sampled at seven sites in the Atlantic, North Sea, and Baltic Sea (Barth JMI et al. (2019) Disentangling structural genomic and behavioural barriers in a sea of connectivity. Mol Ecol 28, 1394–1411).

How to do this activity

• Connect to your Amazon instance via the terminal using SSH.
• Questions or activities are indicated with Q.
• All text in red on gray background indicates commands or file names that you can type or copy to the terminal. Text in black on gray background refers to terminal outputs.
• Try to work independently using the provided manuals and information, discuss questions with your neighbor. If you get stuck, check the answer-box:
  

  Show me the answer!

  Oh no, only if you get really stuck!! First try to find the answer yourself!
  

There are often alternative options and other solutions to the tasks. E.g., whether you prefer cat over less doesn’t matter in most cases – go ahead and use your solution! Don’t hesitate to ask the workshop team if you have questions!

1) The Variant Call Format (VCF)

Q1 Open the terminal and navigate to the analysis folder including the data file for this activity: /home/wpsg/workshop_materials/21_vcf_overview.
What is the file size of cod204.lg05.1.vcf.gz? How many bytes and how many MB does it have?

Q2 What is written in the first ten lines of the VCF file cod204.lg05.1.vcf.gz?

The first line is already telling you that this is indeed a VCF file:
##fileformat=VCFv4.2

These first lines, starting with ##, are part of the “VCF header”, which stores meta-information about the data. If you use zless you can scroll down to see more of the header and also the genotype data, which make up the majority of the file.

There can be a lot of information in the header. Different meta-information lines, e.g. “INFO”, “FILTER”, “FORMAT”, “ALT” describe types of information that are given for every variant in the VCF file. A few examples:

• ##INFO<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes, for each ALT allele, in the same order as listed">: The number of of often the alternative allele is occuring.
• ##FORMAT<ID=GT,Number=1,Type=String,Description="Genotype">: The genotypes coded as allele values separated by / (or | if the data is phased).

The allele values are 0 for the reference allele and 1 for the alternative allele. If there are more alternative alleles the second alternative allele will have the value 2, the third 3, etc. Missing data is denoted by dots ..

• ##contig<ID=LG01,length=28303952> : Part of a list of chromosomes, contigs, or scaffolds of the reference assembly.

For more information about the different tags, have a look at the VCF file format description.

The very last line of the header specifies the column names for the genotype data. The first eight columns are mandatory, follow by “FORMAT” and the sample IDs:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT AVE1409002 AVE1409003 AVE1409004 [...] TVE130512 TVE130513 TVE130514

You may want to edit these header lines to add changes that you did to your VCF file, or you received a VCF file from colleagues and wish to inspect the header in more detail to see what they did.

Q3 Find a command in BCFtools that allows you to separate the header from the large body of genotype data. Save the header in a separate text file named cod204.lg05.1.header.txt.
To find the command, have a look at the BCFtools manual, or just type bcftools on the command line to see the available options.

Q4 What is the file size of cod204.lg05.1.header.txt, compared to the whole VCF file?

Q5 Open the header on the command line in a text editor. Add a new line below the line ##fileformat=VCFv4.2 stating the date of today: ##fileDate=20200121 .
Save the new header as: cod204.lg05.1.header.ed.txt. Use BCFtools to replace (or “rehead”) the VCF file cod204.lg05.1.vcf.gz with the new edited header and save the re-headed VCF as cod204.lg05.1.ed.vcf.gz.
Make sure your changes took effect and your file is still valid by visually inspecting the file and the file size of cod204.lg05.1.ed.vcf.gz.

Tip: Type bcftools to see general options for the program; then use bcftools reheader to see the more specific options for the reheader function.

In the genotype part of the VCF file, one line corresponds to one variant site. Each line starts with the chromosome ID “CHROM”, the position “POS”, and the variant “ID”, and is followed by the bases of the two alleles (“REF” for the reference allele, and “ALT” for the alternative allele(s)). The 6th column (“QUAL”) is the phred-scaled quality score, a measure for how likely it is that this site is indeed a variant. In the next column, “PASS” tells us that the variant passed all filters.

In the “INFO” column, information related to the header info tags for each variant site is given (e.g., AC is the allele count in genotypes for the “ALT” allele(s), AF the allele frequency of the “ALT” allele, and AN is the total number of alleles, etc. We will use some of the “INFO” field specifications later on for hard quality filtering).

The “FORMAT” column specifies the fields and order of the data that is given with each individual for each genotype and the remaining columns list each individual with the values corresponding to the “FORMAT” field. The first “FORMAT” field is always the genotype (GT), the remaining fields are variable, but in our data file, GT is followed by AD (allelic depths for the ref and alt alleles), DP (read depth), GQ (genotype quality), and PL (phred-scaled genotype likelihood):

This is how the first variant of the genotype part of the VCF file looks like:
LG05 338 . A G 93.01 . AC=1;AF=0.0005862;AN=408;BaseQRankSum=0.674;ClippingRankSum=0;DP=8975;ExcessHet=3.0357;FS=0;InbreedingCoeff=-0.0104;MLEAC=1;MLEAF=0.0005862;MQ=56.57;MQRankSum=0;QD=11.63;ReadPosRankSum=1.24;SOR=0.148;AC_Het=1;AC_Hom=0;AC_Hemi=0 GT:AD:DP:GQ:PL 0/0:20,0:20:57:0,57,855 0/0:9,0:9:27:0,27,345 0/0:10,0:10:30:0,30,381 [...] 0/0:7,0:7:21:0,21,248

Q6 How many variant sites are stored in the VCF?

Q7 Examine the first two variant sites in the VCF file and answer the following questions:

A) What kind of variants do you see (e.g., SNP, insertion/deletion)?

B) What is the genotype (GT) of the first individual (AVE1409002) at the first variant site (LG05 338)?

C) What is the genotype read depth (DP, specified as “FMT/DP” to distinguish it from the mean DP “INFO/DP” per variant site) for the first three individuals (AVE1409002, AVE1409003, AVE1409004) at the first variant site (LG05 338)?