Sequencing by hybridization: towards an automated sequencing of one million M13 clones arrayed on membranes

Abstract

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864-well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96-well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13,824 samples on an 8 x 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200-2000 32P-labeled probes comprising 16-256 10-mers having a common 8-mer, 7-mer, or 6-mer in the middle (20 probes per day, each hybridized with 250,000 dots); (v) scoring hybridization signals of 5 million sample-probe pairs per day using storage phosphor plates; and (vi) computing clone order and partial-to-complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.