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Updated: Mar 31, 2026
Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens
Published on: March 8, 2018
Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using
Sarah M Hykin1, Ke Bi2, Jimmy A McGuire1
1Department of Integrative Biology, 3101 Valley Life Sciences Building, University of California, Berkeley, California, United States of America; Museum of Vertebrate Zoology, 3101 Valley Life Sciences Building, University of California, Berkeley, California, United States of America.
High-throughput sequencing can now recover genetic data from old, formalin-fixed herpetological specimens. This method, using liver tissue, enables phylogenetic analysis of valuable museum collections.
Area of Science:
- Molecular Biology
- Genomics
- Zoology
Background:
- Historical natural history collections contain vast numbers of specimens, primarily amphibians and reptiles, preserved without fresh tissue for genetic analysis.
- Extracting and sequencing DNA from formalin-fixed, ethanol-preserved specimens has been challenging due to DNA fragmentation, limiting their use in phylogenetic studies.
- Massively parallel High-Throughput Sequencing (HTS) offers potential for recovering genetic markers from degraded DNA, including that damaged by formalin fixation.
Purpose of the Study:
- To evaluate DNA extraction methods for formalin-fixed specimens of Anolis carolinensis for phylogenetic analysis.
- To assess the feasibility of using HTS to obtain genetic data from historical herpetological specimens.
Main Methods:
- Compared DNA extraction from three tissue types of formalin-fixed Anolis carolinensis specimens.
- Applied High-Throughput Sequencing (HTS) to extracted DNA.
- Utilized bioinformatics for read mapping, genome reconstruction, and phylogenetic placement.
Main Results:
- Sufficient quality DNA was successfully extracted from liver tissue, demonstrating a minimally destructive technique.
- Sequencing was successful only for a more recently collected specimen (~30 years old), suggesting age and preservation conditions are critical.
- Despite high base misincorporation, 27.93% of cleaned reads mapped to the reference genome, enabling complete mitochondrial genome reconstruction and accurate phylogenetic placement.
Conclusions:
- The success of HTS on formalin-fixed specimens depends on the amount of available DNA, influenced by specimen age and preservation.
- This optimized technique significantly enhances the utility of museum collections for genetic research.
- Previously inaccessible formalin-fixed specimens can now be utilized for genetic analysis and phylogenetic studies.
