Megabase sequencing of human genome by ordered-shotgun-sequencing (OSS) strategy

Ellson Y. Chen

doi:10.1117/12.274362

22 May 1997 Megabase sequencing of human genome by ordered-shotgun-sequencing (OSS) strategy

Ellson Y. Chen

Proceedings Volume 2985, Ultrasensitive Biochemical Diagnostics II; (1997) https://doi.org/10.1117/12.274362
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States

Abstract

So far we have used OSS strategy to sequence over 2 megabases DNA in large-insert clones from regions of human X chromosomes with different characteristic levels of GC content. The method starts by randomly fragmenting a BAC, YAC or PAC to 8-12 kb pieces and subcloning those into lambda phage. Insert-ends of these clones are sequenced and overlapped to create a partial map. Complete sequencing is then done on a minimal tiling path of selected subclones, recursively focusing on those at the edges of contigs to facilitate mergers of clones across the entire target. To reduce manual labor, PCR processes have been adapted to prepare sequencing templates throughout the entire operation. The streamlined process can thus lend itself to further automation. The OSS approach is suitable for large- scale genomic sequencing, providing considerable flexibility in the choice of subclones or regions for more or less intensive sequencing. For example, subclones containing contaminating host cell DNA or cloning vector can be recognized and ignored with minimal sequencing effort; regions overlapping a neighboring clone already sequenced need not be redone; and segments containing tandem repeats or long repetitive sequences can be spotted early on and targeted for additional attention.

Citation Download Citation

Ellson Y. Chen "Megabase sequencing of human genome by ordered-shotgun-sequencing (OSS) strategy", Proc. SPIE 2985, Ultrasensitive Biochemical Diagnostics II, (22 May 1997); https://doi.org/10.1117/12.274362

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