Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA

Journal name:
Nature Protocols
Year published:
Published online
Corrected online


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.

At a glance


  1. Schematic overview of single-stranded library preparation, exemplified by one strand of a double-stranded DNA molecule containing a uracil and a single-strand break.
    Figure 1: Schematic overview of single-stranded library preparation, exemplified by one strand of a double-stranded DNA molecule containing a uracil and a single-strand break.

    A, deoxyadenine; Btn, biotin; Pho, phosphate; U, deoxyuracil.

  2. Schematic representation of the qPCR amplification schemes.
    Figure 2: Schematic representation of the qPCR amplification schemes.

Change history

Corrected online 15 May 2013

In the version of this article initially published, the sequence of oligonucleotide CL78 reported in Table 1 as “(AGATCGGAAG[C3Spacer]10[TEG-biotin] (TEG = triethylene glycol spacer))” is incorrect. The correct sequence is “[Phosphate]AGATCGGAAG[C3Spacer]10[TEG-biotin] (TEG = triethylene glycol spacer)”. The error has been corrected in the HTML and PDF versions of the article.


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Author information


  1. Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.

    • Marie-Theres Gansauge &
    • Matthias Meyer


M.-T.G. and M.M. developed the protocol and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Figure 1 (364 KB)

    Sequences of adapters, amplification and sequencing primers. Adapter sequences of a regular single-indexed Illumina multiplex library (as obtained using Illumina's TruSeq DNA sample preparation kit, cat. no. FC-121-2001/2002, or following the protocol provided in ref. 21 of the main text) are shown on top. Libraries prepared from single-stranded DNA differ by a deletion of 5 bp in the P5 adapter. Both library types are compatible with double-indexed sequencing (see ref. 22 of the main text for details). Amplification and sequencing primers are indicated by arrows. Primers with prefix 'IS' are further described in ref. 21 of the main text.

  2. Supplementary Figure 2 (397 KB)

    Characterization of two DNA libraries prepared with and without uracil removal from a Neanderthal DNA extract (panels on the right and left, respectively). A) Fragment length distributions of the amplified libraries obtained from chip electrophoresis using the Bioanalyzer 2100. B) Fragment size distributions obtained from sequencing (the fraction of mapped sequences is indicated by a dotted line). C) Frequency of C to T substitutions around 5′and 3′ends of Neanderthal sequences. D) Average GC-content of Neanderthal sequences as a function of fragment size.

  3. Supplementary Figure 3 (197 KB)

    Quality control of single-stranded adapter oligonucleotide CL78. From two independently synthesized batches of the oligonucleotide, 10 pmol were loaded onto a 10% denaturing polyacrylamide gel, next to a size marker (lane 1; 20/100 ladder). The gel was run for 35 min at 200V and stained with SybrGold dye. While no impurities were detected in the first batch (lane 2), the second batch (lane 3) is dominated by a double-length artifact, representing an extreme example of poor oligonucleotide synthesis quality.

  4. Supplementary Figure 4 (278 KB)

    Determining the optimal cycle number for indexing PCR using the qPCR amplification plots. Shown are the amplification plots obtained from quantifying the libraries prepared in the experiment described in 'Anticipated results'. The saturation phase of PCR starts after cycle 18 (sample libraries, red and blue) and cycle 23 (blank library, green), respectively. Assuming full amplification efficiency (i.e. a doubling of library molecules in each cycle), the optimal cycle number for indexing PCR can be determined as follows by correcting for differences in reaction volumes and the amount of template DNA: (i) qPCR was performed in 25 μl reactions, whereas indexing PCR is performed in 100 μl volume. Thus, 2 cycles should be added to allow for 4 times more end product. (ii) One microliter of a 1:20 library dilution was used for measurement, whereas 24 μl of the library are used for indexing PCR (480 times as much). This corresponds to 8.9 (rounded 9) cycles that should be deducted. Thus, 11 and 16 were estimated to be the optimal cycle numbers for indexing PCR.

  5. Supplementary Table 1 (255 KB)

    Program settings of Cooling-ThermoMixer MKR13 recommended for single-stranded library preparation. The device may also be used to replace the vortexer in bead resuspension steps (use 'short mix' button).

  6. Supplementary Table 2 (331 KB)

    P5 and P7 indexing primers. An identical set of P7 indexing primers was published before in ref. 21 of the main text.

Additional data