Genomics Technologies

Developed by Frederick Sanger in 1977, who won the Nobel Prize for it. Uses chain-terminating dideoxynucleotides (ddNTPs) - analogues of normal dNTPs that lack the 3'-OH group needed for chain extension. When a ddNTP is incorporated, elongation stops, producing a ladder of fragments of different lengths. Capillary electrophoresis separates by size; fluorescent dye colours identify each base.

Despite being 50 years old, Sanger sequencing remains the gold standard for single-variant confirmation in clinical labs. Any pathogenic variant identified by WES or NGS that will influence clinical management must be confirmed by Sanger before reporting.

• Read length: 600–1000 bp (longest of any sequencing method)
• Accuracy: >99.9% raw - the most accurate sequencing available for a single read
• Throughput: 1–96 reads per run (very low - not scalable to whole genomes)
• Cost: ~$5–10 per reaction (cheap per reaction, expensive per genome)
• Turnaround: 1–2 days (primer design + PCR + sequencing)
• Clinical use: confirm WES/NGS findings before reporting; targeted family testing; confirm pathogenic variants in known disease genes
• Limitation: cannot detect CNVs, structural variants, or variants below ~20% allele frequency

# Typical Sanger confirmation workflow
# 1. Design primers flanking the variant (Primer3, NCBI Primer-BLAST)
#    Target: 200-400 bp amplicon; variant in middle third
# 2. PCR amplification from patient DNA
# 3. Cleanup (ExoSAP-IT or gel purification)
# 4. Cycle sequencing with BigDye Terminator v3.1
# 5. Capillary electrophoresis on ABI 3730xl
# 6. Interpret chromatogram with FinchTV or Sequencher

# Example: Confirming BRCA1 c.5266dupC
# Primer F: 5'-CTTACCTGTTTTATGCATTTT-3'
# Primer R: 5'-TTTCATAAGAAAATTTTGAGCAGTT-3'
# Expected: heterozygous insertion peak visible in chromatogram

Next Generation Sequencing (NGS) revolutionised genomics from 2007 onwards by enabling massively parallel sequencing - millions of DNA fragments sequenced simultaneously in a single run. The key innovation was "sequencing by synthesis" combined with bridge amplification on a flow cell surface, generating millions of clusters each reading the same fragment.

The cost of sequencing a human genome dropped from $3 billion (2003) to under $200 (2023), following a trajectory faster than Moore's Law. This democratised genomics, making clinical WES affordable at ₹15,000–30,000 in India today.

Platform Comparison

• Illumina (NovaSeq, NextSeq, MiSeq) - 150–300 bp reads, >99.9% accuracy, most widely used, ~$1/Gb
• MGI/BGI (DNBSEQ) - similar to Illumina, lower cost, used in large population studies
• Ion Torrent (Thermo Fisher) - semiconductor sequencing, fast runs, higher error in homopolymers
• Complete Genomics - proprietary combinatorial probe-anchor synthesis, population-scale

• PacBio HiFi (SMRT) - 15–25 kb reads, >99.9% HiFi accuracy, methylation detection, $10–15/Gb
• Oxford Nanopore (MinION, PromethION) - real-time sequencing, up to Mb-length reads, ~85–98% raw accuracy
• Advantages over short reads: resolves complex SVs, repeats, phasing, full-length transcripts
• Disadvantage: higher error rate in raw reads (Nanopore), higher cost per Gb (PacBio)

• Fragmentation: sonication or enzymatic to target insert size (150–300 bp for WES)
• End-repair & A-tailing: creates blunt ends then adds 3' adenine overhang
• Adapter ligation: adds platform-specific sequences with unique molecular identifiers (UMIs)
• PCR amplification: 8–12 cycles; too many cycles creates duplicates
• Capture (WES): biotinylated probes hybridise to exonic regions, pull down with streptavidin beads
• Size selection: removes adapter dimers; typically 200–400 bp final library size