Ngs Analysis ToolKit

Common File Formats in DNA/RNA Sequencing

This module introduces key file formats used throughout the sequencing data pipeline, from raw reads to variant calling. Understanding these formats is essential for anyone working with next-generation sequencing (NGS) data.

1. FASTQ - Raw Read Format

• Description: Stores raw sequencing reads along with quality scores.
• Type: Text-based
• Structure:
• Fields:
• @ReadID: Identifier for the sequencing read.
• Sequence line: Nucleotide sequence.
• + separator (optional description).
• Quality scores (ASCII-encoded).
• Used In: Raw data from sequencing platforms, input for aligners (e.g., BWA, Bowtie2).

2. SAM - Sequence Alignment/Map

• Description: Human-readable format for aligned reads.
• Type: Text-based
• Structure: -
• Header lines start with @
• Alignment lines include:
  • Read name
  • Flag
  • Reference name
  • Position
  • Mapping quality
  • CIGAR string
  • Mate information
  • Sequence
  • Quality
• Used In: Intermediate results; inspection/debugging of alignments.

3. BAM - Binary Alignment/Map

• Description: Binary, compressed version of SAM.
• Type: Binary
• Benefits:
• Smaller file size
• Faster for analysis and processing
• Used In: Standard format for storing aligned NGS data.

4. CRAM - Compressed Reference-based Format

• Description: Highly compressed alternative to BAM.
• Type: Binary (uses reference-based compression)
• Benefits:
• More storage-efficient than BAM
• Requires access to the reference genome for decompression
• Used In: Large-scale sequencing projects; long-term storage.

5. VCF - Variant Call Format

• Description: Stores genetic variation (SNPs, indels).
• Type: Text-based
• Structure:
• Fields:
• Chromosome
• Position
• Reference/alternate alleles
• Quality metrics
• Genotype information
• Used In: Output of variant callers (e.g., GATK, FreeBayes).

6. BCF - Binary Call Format

• Description: Binary version of VCF.
• Type: Binary
• Benefits:
• Compact
• Faster for computational tasks
• Used In: Efficient analysis and storage of variant data.

Summary Table

Further Reading

• SAM/BAM Format Specification
• VCF/BCF Format Specification
• HTSlib Documentation