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Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA
Abstract
This protocol describes a method for converting short single-stranded and double-stranded DNA into libraries compatible with high-throughput sequencing using Illumina technology. This method has primarily been developed to improve sequence retrieval from ancient DNA, but it is also applicable to the sequencing of short or degraded DNA from other sources, and it can also be used for sequencing oligonucleotides. Single-stranded library preparation is performed by ligating a biotinylated adapter oligonucleotide to the 3' ends of heat-denatured DNA. The resulting strands are then immobilized on streptavidin-coated beads and copied with a polymerase. A second adapter is attached by blunt-end ligation, and library preparation is completed by PCR amplification. We estimate that intact DNA strands are recovered in the library with ∼50% efficiency. Libraries can be generated from up to 12 DNA or oligonucleotide samples in parallel within 2 d.
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If prepared correctly, immortal DNA sequencing library can be theoretically reamplified indefinitely without exhaustion or loss of complexity.
“If prepared correctly, this library can be theoretically reamplified indefinitely without exhaustion or loss of complexity 143 .”
From Ancient DNA analysis (2021) · cites this paper as [143]
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“When using single-strand library protocols [ 7 , 8 ], only the C-to-T substitutions will be observed.”
Metadata
- Authors
- Gansauge, M.-T., & Meyer, M.
- Affiliations
- Max Planck Institute for Evolutionary Anthropology (DE)
- Max Planck Society (DE)
- Corresponding author
- Gansauge, Marie-Theres
- Meyer, Matthias
- Venue
- Nature Protocols
- Published
- 2013-03-14
- Volume
- 8
- Issue
- 4
- Pages
- 737-748
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2010018187
- Cited by
- 561
- References
- 7
- Citations by year
- 2026: 42 · 2025: 39 · 2024: 39 · 2023: 42 · 2022: 41 · 2021: 53 · 2020: 59 · 2019: 45 · 2018: 68 · 2017: 39 · 2016: 37 · 2015: 31 · 2014: 20 · 2013: 5
- FWCI
- 10.23
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Keywords
- single-stranded DNA, ancient DNA, degraded DNA, DNA library preparation, streptavidin-coated beads, oligonucleotide sequencing, next-generation sequencing
- MeSH terms
- DNA, Single-Stranded, Gene Library, Polymerase Chain Reaction, Sequence Analysis, DNA, DNA Primers, Biotinylation, High-Throughput Nucleotide Sequencing
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