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Representation of cloned genomic sequences in two sequencing vectors: correlation of DNA sequence and subclone distribution.

ABSTRACT: Representation of subcloned Caenorhabditis elegans and human DNA sequences in both M13 and pUC sequencing vectors was determined in the context of large scale genomic sequencing. In many cases, regions of subclone under-representation correlated with the occurrence of repeat sequences, and in some cases the under-representation was orientation specific. Factors which affected subclone representation included the nature and complexity of the repeat sequence, as well as the length of the repeat region. In some but not all cases, notable differences between the M13 and pUC subclone distributions existed. However, in all regions lacking one type of subclone (either M13 or pUC), an alternate subclone was identified in at least one orientation. This suggests that complementary use of M13 and pUC subclones would provide the most comprehensive subclone coverage of a given genomic sequence.

SUBMITTER: Chissoe SL

PROVIDER: S-EPMC146865 | biostudies-other | 1997 Aug

REPOSITORIES: biostudies-other

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Representation of cloned genomic sequences in two sequencing vectors: correlation of DNA sequence and subclone distribution.

Chissoe S L SL Marra M A MA Hillier L L Brinkman R R Wilson R K RK Waterston R H RH

Nucleic acids research 19970801 15

Representation of subcloned Caenorhabditis elegans and human DNA sequences in both M13 and pUC sequencing vectors was determined in the context of large scale genomic sequencing. In many cases, regions of subclone under-representation correlated with the occurrence of repeat sequences, and in some cases the under-representation was orientation specific. Factors which affected subclone representation included the nature and complexity of the repeat sequence, as well as the length of the repeat re ...[more]

PMID: 9224593

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Project description:Mutagenesis in combination with Genotyping by Sequencing (GBS) is a powerful tool for introducing variation, studying gene function and identifying causal mutations underlying phenotypes of interest in crop plant genomes. About 400 million paired-end reads were obtained from 82 ethylmethane sulfonate (EMS) induced mutants and 14 wild-type accessions of Jatropha curcas for the detection of Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletions (InDels) by two different approaches (nGBS and ddGBS) on an Illumina HiSeq 2000 sequencer. Using bioinformatics analyses, 1,452 induced SNPs and InDels were identified in coding regions, which were distributed across 995 genes. The predominantly observed mutations were G/C to A/T transitions (64%), while transversions were observed at a lower frequency (36%). Regarding the effect of mutations on gene function, 18% of the mutations were located in intergenic regions. In fact, mutants with the highest number of heterozygous SNPs were found in samples treated with 0.8% EMS for 3 h. Reconstruction of the metabolic pathways showed that in total 16 SNPs were located in six KEGG pathways by nGBS and two pathways by ddGBS. The most highly represented pathways were ether-lipid metabolism and glycerophospholipid metabolism, followed by starch and sucrose metabolism by nGBS and triterpenoid biosynthesis as well as steroid biosynthesis by ddGBS. Furthermore, high genome methylation was observed in J. curcas, which might help to understand the plasticity of the Jatropha genome in response to environmental factors. At last, the results showed that continuously vegetatively propagated tissue is a fast, efficient and accurate method to dissolve chimeras, especially for long-lived plants like J. curcas. Obtained data showed that allelic variations and in silico analyses of gene functions (gene function prediction), which control important traits, could be identified in mutant populations using nGBS and ddGBS. However, the handling of GBS data is more difficult and more challenging than the traditional TILLING strategy in mutated plants, since the Jatropha genome sequence is incomplete, which makes alignment and variant analysis of target sequence reads challenging to perform and interpret. Therefore, providing a complete Jatropha reference genome sequence with high quality should be a priority for any breeding program.

Dataset Information

Representation of cloned genomic sequences in two sequencing vectors: correlation of DNA sequence and subclone distribution.

Publications

Representation of cloned genomic sequences in two sequencing vectors: correlation of DNA sequence and subclone distribution.

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