Long-range genomic enrichment, sequencing, and assembly to determine unknown sequences flanking a known microRNA

Zhaorong Ma, Michael Axtell

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Conserved plant microRNAs (miRNAs) modulate important biological processes but little is known about conserved cis-regulatory elements (CREs) surrounding MIRNA genes. We developed a solution-based targeted genomic enrichment methodology to capture, enrich, and sequence flanking genomic regions surrounding conserved MIRNA genes with a locked-nucleic acid (LNA)-modified, biotinylated probe complementary to the mature miRNA sequence. Genomic DNA bound by the probe is captured by streptavidin-coated magnetic beads, amplified, sequenced and assembled de novo to obtain genomic DNA sequences flanking MIRNA locus of interest. We demonstrate the sensitivity and specificity of this enrichment methodology in Arabidopsis thaliana to enrich targeted regions spanning 10-20 kb surrounding known MIR166 and MIR165 loci. Assembly of the sequencing reads successfully recovered all targeted loci. While further optimization for larger, more complex genomes is needed, this method may enable determination of flanking genomic DNA sequence surrounding a known core (like a conserved mature miRNA) from multiple species that currently don't have a full genome assembly available.

Original languageEnglish (US)
Article numbere83721
JournalPloS one
Volume8
Issue number12
DOIs
StatePublished - Dec 20 2013

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MicroRNAs
microRNA
Genes
genomics
DNA sequences
Genome
Biological Phenomena
Streptavidin
DNA Probes
loci
Arabidopsis
genome assembly
nucleotide sequences
streptavidin
regulatory sequences
Sensitivity and Specificity
nucleic acids
genes
Arabidopsis thaliana
methodology

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

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abstract = "Conserved plant microRNAs (miRNAs) modulate important biological processes but little is known about conserved cis-regulatory elements (CREs) surrounding MIRNA genes. We developed a solution-based targeted genomic enrichment methodology to capture, enrich, and sequence flanking genomic regions surrounding conserved MIRNA genes with a locked-nucleic acid (LNA)-modified, biotinylated probe complementary to the mature miRNA sequence. Genomic DNA bound by the probe is captured by streptavidin-coated magnetic beads, amplified, sequenced and assembled de novo to obtain genomic DNA sequences flanking MIRNA locus of interest. We demonstrate the sensitivity and specificity of this enrichment methodology in Arabidopsis thaliana to enrich targeted regions spanning 10-20 kb surrounding known MIR166 and MIR165 loci. Assembly of the sequencing reads successfully recovered all targeted loci. While further optimization for larger, more complex genomes is needed, this method may enable determination of flanking genomic DNA sequence surrounding a known core (like a conserved mature miRNA) from multiple species that currently don't have a full genome assembly available.",
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Long-range genomic enrichment, sequencing, and assembly to determine unknown sequences flanking a known microRNA. / Ma, Zhaorong; Axtell, Michael.

In: PloS one, Vol. 8, No. 12, e83721, 20.12.2013.

Research output: Contribution to journalArticle

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