title: Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.
abstract: Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.
authors: Sequencing Research and Development, Wellcome Trust Sanger Institute, Cambridge, UK.
authors: Mike A
authors: MA
authors: Quail
authors: Lucy
authors: L
authors: Matthews
authors: Sarah
authors: S
authors: Sims
authors: Christine
authors: C
authors: Lloyd
authors: Helen
authors: H
authors: Beasley
authors: Simon W
authors: SW
authors: Baxter
mesh_terms: D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D030541:Databases, Genetic; D005822:Genetic Vectors / Q000235:genetics; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA