Sequencing-by-Synthesis (SBS)
Definition
The Illumina detection paradigm: reversibly fluorescent 3'-blocked nucleotides are incorporated one cycle at a time into clonally amplified clusters on a flow cell; laser imaging at each cycle reads the incorporated base by its colour. SBS generates short reads (150-300 bp) at high throughput, distinguishing it from Sanger's long-read (700-1000 bp) single-molecule extension.
- Vendor paradigm
- Illumina
- Chemistry
- Reversibly fluorescent 3'-blocked nucleotides
- Substrate
- Clonally amplified clusters on a flow cell
- Read length
- 150 to 300 bp
- Contrast with Sanger
- Short high-throughput reads vs Sanger's 700-1000 bp single-molecule reads
Common questions
Why does SBS use reversibly blocked nucleotides instead of the natural kind?+
The 3' blocking group stops the growing strand after each single base is added, allowing the instrument to image and identify that one base by its fluorescent colour before the block is chemically removed to allow the next cycle, keeping every cluster synchronised one base at a time.
What is the main forensic advantage SBS offers over Sanger sequencing for DNA casework?+
SBS can sequence millions of DNA fragments in parallel on one flow cell, which underlies massively parallel sequencing approaches used for expanded STR and SNP panels, mitochondrial genome sequencing, and mixture deconvolution that would be impractical one fragment at a time with Sanger.
Why is Sanger sequencing still preferred for some forensic applications despite SBS's throughput advantage?+
Sanger's longer read length and lower per-sample complexity make it well suited to confirming a single specific region, such as verifying a mitochondrial DNA variant or a single-locus result, without the bioinformatics overhead that short-read SBS data requires.
Related terms
- 2',3'-Dideoxynucleotide Triphosphate (ddNTP)
- A nucleotide analogue lacking the 3'-hydroxyl group present in normal dNTPs. When incorporated by DNA polymerase into a growing chain, it prevents...
- Big Dye Terminator (BDT) V3.1
- Applied Biosystems' (now Thermo Fisher Scientific's) commercial cycle sequencing kit using four spectrally distinct dichlororhodamine dyes coupled to the four ddNTPs. The...
- Cycle Sequencing
- A Sanger sequencing format using a thermostable DNA polymerase and repeated denaturation-annealing-extension cycles (typically 25-35 cycles). Produces exponentially more termination products from...
- EMPOP (ENBISI Mitochondrial Population Database)
- The ENFSI-curated reference database of forensic-grade mtDNA haplotypes used to estimate the frequency of a given control-region profile in a specified population....
- Phred Quality Score
- A logarithmic estimate of base-calling error probability (Phred Q = -10 log10 P). Q20 = 1% error rate; Q30 = 0.1% error...
- rCRS (Revised Cambridge Reference Sequence)
- The standard reference sequence for the human mitochondrial genome (NC_012920.1, 16,569 bp), used as the coordinate system for reporting all forensic mtDNA...