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Artifactual pyrosequencing reads in multiple-displacement-amplified sediment metagenomes from the Red Sea

Yong Wang1, On On Lee, Jiang Ke Yang

  • 1Division of Life Science, Hong Kong University of Science and Technology , Hong Kong SAR , China.

Peerj
|May 7, 2013
PubMed

Insights

Multiple Displacement Amplification (MDA) can create artifacts in DNA samples. This study found artifactual gene fragments and stem-loop structures in Red Sea sediment DNA amplified using MDA.

Area of Science:

  • Genomics
  • Molecular Biology
  • Marine Science

Background:

  • The Multiple Displacement Amplification (MDA) protocol is widely used for whole-genome amplification.
  • MDA is known to introduce artifacts, especially in samples with impurities.
  • Sediment DNA from extreme environments like deep-sea brine basins can be challenging for accurate sequencing.

Purpose of the Study:

  • To investigate artifact formation during MDA of deep-sea sediment DNA.
  • To characterize the nature and potential origin of MDA-induced artifacts.
  • To assess the impact of MDA on metagenomic data from Red Sea brine basins.

Main Methods:

  • DNA extraction from Red Sea deep-sea sediment samples.
  • Whole-genome amplification using the MDA protocol.
  • Metagenomic sequencing using 454 pyrosequencing.
  • Bioinformatic analysis to identify artifactual reads and structures.

Main Results:

  • Abundant artifactual reads (50-220 bp) were detected in MDA-amplified metagenomes.
  • These reads contained translocated gene fragments, sometimes from different species.
  • Over 60% of artifactual reads exhibited strong stem-loop secondary structures.

Conclusions:

  • MDA amplification is likely responsible for generating artifactual reads with secondary structures in deep-sea sediment metagenomes.
  • Translocation of gene fragments and subsequent secondary structure formation are key MDA-induced artifacts.
  • Understanding these artifacts is crucial for accurate metagenomic analysis of environmental DNA.
Keywords:
Artifactual 454 readsGene fragmentsMDAMetagenome

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