Use of transcriptional sequencing in difficult to read areas of the genome.
In genome and cDNA sequencing projects, current cycle sequencing often encounters difficult-to-sequence templates which have unique secondary structures due to GC-rich composition or repeated regions. Due to the formation of stable secondary structures, remarkable decreases in fluorescent signals are observed in cycle sequencing reactions. It is not easy to determine the nucleotide sequences of these regions. Although several modifications of sequencing reactions have been tried to overcome these problems, some unreadable regions remain as gaps in genome sequencing projects. Here, we further developed transcriptional sequencing technology and evaluated the sequencing accuracy in these regions. The method was successively applied to artificial GC cluster templates and putative secondary structure-forming templates from genomic and cDNA clones. Our results indicate that transcriptional sequencing is a powerful and accurate method for GC-rich regions, simple sequence repeats, hairpins (inverted repeats), tandem repeat DNA templates, and gap-closing in draft sequencing data.
Ishikawa
Tomokazu
T
Genome Research Laboratories, Wako Pure Chemical Industries, Ltd., 6-1, Takada-cho, Amagasaki, Hyogo 661-0963, Japan.
Hayashida
Yukinobu
Y
Hirayasu
Kazunari
K
Ozawa
Kaori
K
Yamamoto
Naoyuki
N
Tanaka
Takumi
T
Matsuura
Shuji
S
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D016678:Genome; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics*; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase