2 base encoding
These technologies generate hundreds of thousands of small sequence reads at one time.
[1] However, instead of using fluorescent labeled 9-mer probes that distinguish only 6 bases, 2-base encoding takes advantage of fluorescent labeled 8-mer probes that distinguish the two 3 prime most bases but can be cycled similar to the Macevicz method, thus greater than 6bp reads can be obtained (25-50bp published,[2] 50bp in NCBI in Feb 2008).
[3][4][5][6] The general steps common to many of these next-generation sequencing techniques include: In 1988, Whiteley et al. demonstrated the use of fluorescently labeled oligonucleotide ligation for the detection of DNA variants.
[9] In 1995 Macevicz[10] demonstrated repeated ligation of oligonucleotides to detect contiguous DNA variants.
In 2003, Dressman et al.[11] demonstrated the use of emulsion PCR to generate millions of clonally amplified beads which one could perform these repeated ligation assays on.
The resulting library contains template DNA fragments, which are tagged with one adapter at each end (A1-template-A2).
- Step 2, Emulsion PCR: In this step, the emulsion (droplets of water suspended in oil) PCR reaction is performed using DNA fragments from library, two primers (P1 and P2) that complement to the previously used adapters (P1 with A1 and P2 with A2), other PCR reaction components and 1μm beads coupled with one of the primers (e.g. P1) make dilution from DNA library to maximize the droplet that contain one DNA fragment and one bead into a single emulsion droplet.
Then DNA polymerase will extend from P1 to make the complementary sequence, which eventually results in a bead enriched with PCR products from a single template.
After enrichment, the 3’-end of products (P2 end) will be modified which makes them capable of covalent bonding in the next step.
Cleavage of the fluorescent dye and bases 6-8 leaves a free 5' phosphate group ready for further ligation.
The remaining three rounds will be performed with new universal primers annealing positions n-2, n-3 and n-4 relative to the 3'-end of P1.
Direct decoding or translation of the color reads into bases cannot do this efficiently without other knowledge.
When base calling single color miscalls cause errors on the remaining portion of the read.
However for simplistic de novo assembly you are left with the raw device error rate which will be significantly higher than the 0.06% reported for SNP calling.
