SNP Calling: A Step-by-Step Guide

SNP Calling refers to the process of identifying single nucleotide polymorphisms (SNPs) in sequencing data, distinguishing genuine variations from sequencing errors. Here’s a step-by-step guide to perform SNP calling using bioinformatics tools and scripts:

Step 1: Prepare Your Data

1. Obtain Sequence Data: Ensure you have quality-checked FASTQ files containing your sequencing reads.
2. Reference Genome: Download the reference genome for your organism of interest (e.g., from NCBI or Ensembl).

Step 2: Align Reads to the Reference Genome

Use an aligner like BWA or Bowtie2 to map reads to the reference genome:

Convert the SAM file to BAM, sort, and index:

Step 3: Mark Duplicates (Optional)

Use Picard Tools to mark PCR duplicates:

Step 4: Call Variants

Use a variant caller like GATK HaplotypeCaller:

Alternatively, use BCFtools:

Step 5: Filter Variants

Filter SNPs based on quality metrics:

Step 6: Annotate Variants

Annotate SNPs with functional information using SnpEff or ANNOVAR:

Recent Online Tools and Software for SNP Calling

• GATK (Genome Analysis Toolkit): Comprehensive tool for variant calling and filtering.
• BCFtools: Lightweight and efficient variant calling tool.
• FreeBayes: Suitable for pooled sequencing or polyploid genomes.
• VarScan: Focused on high-confidence SNP and indel detection.
• DeepVariant: Uses deep learning for variant calling.

Example Python Script for Simple SNP Calling (using Pysam)

Conclusion

SNP calling involves aligning reads, identifying variants, and filtering them for accuracy. Tools like GATK, BCFtools, and DeepVariant offer robust solutions. Combining computational methods with biological interpretation ensures high-confidence SNP discovery.