"Next Generation Sequencing"

Sanger Sequencing by means of dideoxy terminators was the method of choice for manual and automated DNA sequencing for fifty years, an extraordinarily long time for a biotechnology. New methods of "Next Generation Sequencing" began to be developed in the 90s, and are now routine in the 21st century [the name is a nod to 'Star Trek: the Next Generation' as a sequel to The Original Series (TOS), perhaps The Original Sequences ?].

    In the NGS method illustrated, (1) Fragments of a particular DNA molecule of interest are denatured into single-stranded DNA (ssDNA). The 5' and 3' ends of the ssDNA fragments are tagged with short single-stranded adapter sequences (2) The fragment adapters are "tethered" to a dense carpet of identical base-complementary 5' & 3' adapters on a matrix in a DNA Flow cell. The Flow cell over allows different reaction mixtures to be passed over the matrix during the sequencing process. (3) An adapted ssDNA fragment attached at the 5' end 'bends over' and attaches to a nearby 3' adapter as a "bridge". (4) Each bridge is amplified by PCR in the usual 5' $→$3' manner. The bridge is now double-stranded. (5) Denaturation of the bridge now leaves two single strands attached to the matrix, one the original fragment in Step 2, and the other its complement. (6) From these two complementary strands, each repetition of Steps 3 - 5 doubles the number of single stranded templates in each cycle.

    [Bottom, left] The original DNA fragments are widely spaced, so that the amplified fragments for each occurs cluster in a small area of the matrix. [Middle] A sequencing reaction is performed over the entire matrix simultaneously, adding one nucleotide at a time. [right] Each addition is scanned in succession, so as to build up  the sequences of all original fragments. The sequences obtained in this manner are relatively short, but are sufficiently long that a computer can assemble overlapping short sequences as a single composite sequence of the entire molecule of interest.