Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects

Analysis of the 3'-ends of approximately 900 separate human LINE-1 (L1) elements from primates revealed 47 contiguous but distinct subfamilies with the L1 family. Eight previously described medium reiteration frequency sequences (MERs) were found to be parts of ancient L1 untranslated 3'-regions which show little or no sequence similarity to the presently active L1 3'-end. Some of the major changes in 3'-end sequence can be explained by recombination events between different L1 repeats as well as between L1 and unrelated repetitive sequences. One of these sequences, MER42, is reported in this paper. With the set of consensus sequences for different subfamilies and their diagnostic features, it is possible to estimate the age of individual LINE-1 elements. Contrary to earlier suggestions, the majority of L1 copies in the human genome is very old; more than half of the identifiable elements were inserted into the genome before the mammalian radiation, as evidenced by elements at orthologous sites in human and other mammalian genomes. Multiple distinct L1 source genes seem to have been active simultaneously over long periods of time.

A mismatch-free hybridization of oligonucleotides containing from 11 to 20 monomers to unknown DNA represents, in essence, a sequencing of a complementary target. Realizing this, we have used probability calculations and, in part, computer simulations to estimate the types and numbers of oligonucleotides that would have to be synthesized in order to sequence a megabase plus segment of DNA. We estimate that 95,000 specific mixes of 11-mers, mainly of the 5'(A,T,C,G)(A,T,C,G)N8(A,T,C,G)3' type, hybridized consecutively to dot blots of cloned genomic DNA fragments would provide primary data for the sequence assembly. An optimal mixture of representative libraries in M13 vector, having inserts of (i) 7 kb, (ii) 0.5 kb genomic fragments randomly ligated in up to 10-kb inserts, and (iii) tandem "jumping" fragments 100 kb apart in the genome, will be needed. To sequence each million base pairs of DNA, one would need hybridization data from about 2100 separate hybridization sample dots. Inevitable gaps and uncertainties in alignment of sequenced fragments arising from nonrandom or repetitive sequence organization of complex genomes and difficulties in cloning "poisonous" sequences in Escherichia coli, inherent to large sequencing by any method, have been considered and minimized by choice of libraries and number of subclones used for hybridization. Because it is based on simpler biochemical procedures, our method is inherently easier to automate than existing sequencing methods. The sequence can be derived from simple primary data only by extensive computing. Phased experimental tests and computer simulations increasing in complexity are needed before accurate estimates can be made in terms of cost and speed of sequencing by the new approach. Nevertheless, sequencing by hybridization should show advantages over existing methods because of the inherent redundancy and parallelism in its data gathering.