Tools and Methods to Analyze Variant and Genotyping of Human Genome Data 12/1/2021

Recently I’ve been tasked with analyzing Biological and Genomic data. I’ve learned a lot about tools and libraries for R and Python. As part of this analysis, I’m helping scientists analyze genome variations and perform analysis of genotypes. The human genome has 23 chromosomes. That’s about 3 billion base pairs that contain around 30,000 genes. Every base has pair that can be coded with 2 bits. This equates to around 750 megabytes of data. The data that I have been analyzing is several terabytes of genome sequences for around eight humans. Because the data is so massive, there are several high-throughput tools available to perform genotyping and variation discovery that I will cover in a series of articles in the next few months.

One characteristic is that repetitive DNA sequences comprise approximately 50% of the human genome. Genome size 3,100 Mbps (mega-basepairs) per haploid genome. A base pair is two chemical bases bonded to one another forming the “rung” in the DNA. DNA strands look someone like ladder twisted around.


Variations include differences in the number of copies individuals have of a particular gene, deletions, translocations and inversions. One such variation is Single-nucleotide polymorphism (SNPs). It’s a type of copy number variation (CNV), Variations in DNA are actually a normal part of human genetics and can sometimes be a sign of the body adapting to various changes within the sequences or even adaptation for protecting and adapting.

SNP can be any nucleic acid substitution:

  1. Transition
    1. Interchange of the purine (Adenine/Guanine)
    2. Pyrimidine (Cytosine/Thymine) nucleic acids
  2. Transversion
    1. Interchange of purine and pyrimidine nucleic acid

Since variation discovery is very important in the biological sciences, many tools have been developed to assist in creating medicines and treatments for all type of mutations within cells.

The International HapMap Project was develop a describe of variation patterns in the human genome that finds variations that impact health, responses to drugs and an individual’s environment. Variations include small-scale and large-scale variations.

Copy number variation (CNV). With the number of copies of a particular gene varies from one individual to the next. Following the completion of he Human Genome Project, it became apparent that the genome experiences gains and losses of genetic material. The extent to which copy number variation contributes to human disease is not yet known. It has long been recognized that some cancers are associated with elevated copy numbers of particular genes. They are categorized as long repeats or short repeats.

Insertions and Deletions (InDel) are a type of CNV: Insertion-deletion mutations refer to insertion and/or deletion of nucleotides into genomic DNA and include events less that 1Kb in length.

Other Definitions

Length of the base pairs (bp). One bp corresponds to approximately 3.4 A (340 pm) of length along the strand, and to roughly 618 or 643 daltons for DNA and RNA respectively.

Kilobase (kb) is a unit of measurement in molecular biology equal to 1000 base pairs of DNA or RNA.

Data Analysis

Most of analysis is performed in R. Here are some of the analysis done using Genome libraries:

# prepare for transaction data


The above libraries are standard R libraries for analyzing Genomic data. Later in this document, I will discuss the multiple tools that produce the files necessary for these libraries.

The most advanced libraries can be downloaded from the BiocManager website.

if (!require("BiocManager", quietly = TRUE))

if (!requireNamespace("BiocManager", quietly = TRUE))





Using R with Human Genome Variation Data

Reading VCF files into R.

The vcfR package was designed to work with data from VCF files. The vcfR package was designed to work on an individual chromosome. A VCF file structure is a standard file format for storing variations for genomic data and is used by organizations to map human genome variations. It used used for large scale variant mapping. One example is the International Genome Sample Resource (IGSR).

It contains headers

  1. CHROM
  2. POS
  3. ID
  4. REF
  5. ALT
  6. QUAL
  8. INFO
  1. The name of the chromosome.
  2. The starting position of the variant indicated.
  3. Identifier
  4. Reference allele. An allele is one of two or more alternative forms of a gene that occur by mutation and found in the same area of a chromosome.
  5. Alternate allele
  6. Quality score out of 100.
  7. Pass/Fail. Did it pace quality filters.
  8. Information about the following columns.
  9. Format of the columns.

FASTQ file format

The FASTQ files contain entire genome sequencing and can be very large and represents the raw sequencing data.

BAM or CRAM file formats

These are the files that align sequencing data with referencing genome data.

Genomics Tools

Genome data is very large, and contains millions of base pairs for chromosomes. Although this data can be loaded into R, the complexity of looking at individual genes and chromosomes can be very daunting. One tool that makes reading genomic data more visual is Integrative Genomics Viewer or (IGV). IVG is a visualization tool that zooms in to the gene and chromosome level at the base length.

IGV efficiently pulls in BAM file indexes to locate genomic data.

Other tools for Genomics, structural biology and molecular biology is DNASTAR Lasergene Structural Biology Suite and Spartan.

Another tool for visualization is the Variant Effect Predictor (VEP), which determines the effect of variants on genes.

Other tools include SnpEff and SnpSuft. SnpEff provides genetic variant annotation and function effect prediction. It also annotates and predicts the effect of genetic variants on genetic variants on genes and protein.

SnpSift annotates genomic variants using database, filters, and annotated variants. Once you annotated your files using SnpEff, you can use SnpFift to help you filter large genomic datasets in order to find the most significant variants for your experiment. Microsoft Genomics: All SnpEff & SnpSift genomic database are kindly hosted by Microsoft Genomics and Azure

Microsoft Genomics service provides a cloud hosted solution that makes it easy to variant call your genomic samples. The service takes in genomic samples as two paired end read fastq (.fq.gz) files and produces .bam, .bai, or.vcf files, along with the associated log files.

The process uses a BWA / GATK data pipeline where Microsoft has improve the efficiency of both BWA and GATK producing results faster and with less overhead. There is also a secondary analysis .

GATK is the Genome Analysis Toolkit also used for variant discovery using a data pipeline which can be scaled in the Azure or Google cloud. GATK is a framework for Variant Discovery with high-throughput sequencing data.

Another tool is the CNVnator is a tool for CNV discovery and genotyping from depth-of-coverage by mapped reads


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