title: Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing.
abstract: Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.
authors.affiliation: Science for Life Laboratory, KTH, Gene Technology, Solna, Sweden.
authors.forename: Sverker
authors.initials: S
authors.lastname: Lundin
authors.forename: Joel
authors.initials: J
authors.lastname: Gruselius
authors.forename: Björn
authors.initials: B
authors.lastname: Nystedt
authors.forename: Preben
authors.initials: P
authors.lastname: Lexow
authors.forename: Max
authors.initials: M
authors.lastname: Käller
authors.forename: Joakim
authors.initials: J
authors.lastname: Lundeberg
mesh_terms: D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D045744:Cell Line, Tumor; D058893:DNA Barcoding, Taxonomic; D004285:Dogs / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008928:Mitochondria / Q000235:genetics*; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D009841:Oligonucleotides; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53