Dataset Information

Comparison and optimization for DNA extraction of archived fish specimens.

ABSTRACT: The DNA extracted from museum alcohol-fixed specimens can be a valuable source of information for solving taxonomic, phylogenetic, ecological and conservational questions. However, this type of DNA, also called ancient DNA, is routinely obtained in small portions and highly fragmented. We have tested two different extraction kits in museum type-specimens of the fish family Characidae. Aiming to increase the DNA yield, we made modifications on a Qiagen manufacturer protocol, in the elution step. Also, to overcome the issue of DNA fragmentation, we applied our efforts in Sanger sequencing, to find a highly variable and, in result, informative COI fragment. Based on our results, there is no correlation between amount of the DNA extracted and the age of the sample. The Sanger sequencing generated sequences which are useful in solving taxonomic puzzles. Here are presented the customization and guidelines that allowed us to recover DNA from the archived fish specimens. •DNA extraction from archived fish specimens is more effective when using silica columns.•Change of the elution times from minutes in room temperature to 24?h in freezer greatly improved the DNA yielded.•Short but highly variable sequences replace the need to sequence the entire gene to identify a species.

SUBMITTER: Silva PC

PROVIDER: S-EPMC6584492 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Comparison and optimization for DNA extraction of archived fish specimens.

Silva Priscilla C PC Malabarba Maria Claudia MC Vari In Memoriam Richard R Malabarba Luiz R LR

MethodsX 20190608

The DNA extracted from museum alcohol-fixed specimens can be a valuable source of information for solving taxonomic, phylogenetic, ecological and conservational questions. However, this type of DNA, also called ancient DNA, is routinely obtained in small portions and highly fragmented. We have tested two different extraction kits in museum type-specimens of the fish family Characidae. Aiming to increase the DNA yield, we made modifications on a Qiagen manufacturer protocol, in the elution step. ...[more]

PMID: 31249793

Similar Datasets

Project description:Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish.•A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method.•The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications.•The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.

Project description:DNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure.We designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments.In phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the UltraClean® Tissue and Cells treatments, a result likely arising from using a combination of different beads during homogenization.Both the PowerBiofilm® and UltraClean® Tissue and Cells treatments are appropriate for large-scale analyses of coral microbiota. However, studies interested in detecting cryptic microbial members may benefit from using the PowerBiofilm® DNA treatment because of the likely enhanced lysis efficiency of microbial cells attributed to using a variety of beads during homogenization. Consideration of the methodology involved with microbial DNA extraction is particularly important for studies investigating complex host-associated microbiota.

Dataset Information

Comparison and optimization for DNA extraction of archived fish specimens.

Publications

Comparison and optimization for DNA extraction of archived fish specimens.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets