An optimized method for the extraction of ancient eukaryote DNA from marine sediments

Area of Science:

• Marine biology
• Molecular ecology
• Paleogenomics

Background:

• Marine sedimentary ancient DNA (sedaDNA) is crucial for reconstructing past marine food webs.
• Existing sedaDNA extraction protocols often struggle to efficiently recover small DNA fragments characteristic of ancient DNA (aDNA).
• A need exists for optimized protocols to maximize eukaryotic DNA yield from marine sediments.

Purpose of the Study:

• To compare seven sedaDNA extraction and library preparation methods.
• To identify an optimized protocol for maximizing sedaDNA yield from marine sediments.
• To improve the detection of highly fragmented eukaryotic DNA in sediments.

Main Methods:

• Compared seven sedaDNA extraction treatments and library preparations on marine sediment samples.
• Tested variables including sediment storage (frozen vs. refrigerated), cell lysis (bead-beating vs. EDTA), DNA binding (silica spin column vs. silica-solution), library preparation (diluted vs. undiluted DNA), and size-selection.
• Applied a stringent bioinformatic filtering approach for metagenomic data.

Main Results:

• Maximum sedaDNA extraction efficiency was achieved using frozen sediments, EDTA incubation, bead-beating, DNA binding in silica-solution, and undiluted DNA in shotgun libraries.
• The optimized protocol successfully identified 45 marine eukaryotic taxa.
• An optional low molecular-weight (LMW) size-selection step was effective for retaining DNA fragments ≤500 base pairs.

Conclusions:

• An optimized sedaDNA extraction and data-processing protocol was developed, integrating EDTA incubation, bead-beating, silica-solution binding, and specific library preparation steps.
• This protocol enhances the quantitative paleo-monitoring of marine eukaryotes and the detection of degraded DNA in sediments.
• The study provides a valuable reference list of contaminants for future sedaDNA research.