Library Preparation

We prepared libraries using the SCR, BEST, and ssDNA2.0 library preparation protocols (Figure 1). To assess whether library performance varied by DNA input amounts, we first prepared SCR, BEST, and ssDNA2.0 libraries from the extraction PH158 using 6 different inputs: 1.00 pmols (29.70 ng), 0.50 pmols (14.85 ng), 0.25 pmols (7.43 ng), 0.125 pmols (3.71 ng), 0.063 pmols (1.85 ng), and 0.032 pmols (0.93 ng) of ssDNA or dsDNA ends. Next, we assessed library performance consistency among samples by preparing SCR, BEST, and ssDNA2.0 libraries from each of the 4 remaining DNA extracts using an input of 0.125 pmols (3.71 ng) of DNA. All final pre-amplified libraries were eluted in 50 uL of EBT buffer.

Below, we describe briefly the 3 library preparation protocols. A detailed description of the SCR is provided as supplementary material.

The BEST protocol (Figure 1A) is a single-tube double-stranded DNA (dsDNA) library preparation protocol optimized for ancient DNA. We prepared BEST libraries as outlined in Carøe et al. (Carøe et al. 2017) with the modifications described in Mak et al. (Mak et al. 2017), using a 25:1 adapter:template ratio. We used a MinElute column for the final clean-up prior to amplification.

Briefly, BEST libraries are prepared by first performing an end-repair reaction with T4 Polynucleotide Kinase (NEB) and T4 DNA Polymerase (NEB) which blunt-ends the input DNA. Following end-repair, a blunt-end ligation reaction is performed using T4 DNA Ligase (NEB) which facilitates the ligation of the 5′ ends of template molecules to the 3′ ends of blunt-end adapters. Then, an adapter fill-in reaction is performed with Bst 2.0 DNA Polymerase (NEB), which initiates at the ligation junction nick present at the 3′ ends of the template and 5′ ends of the non-ligated adapter strand. Heat inactivation of enzymes occurs between reactions, but a MinElute (Qiagen, Hilden, Germany) column clean-up step is performed following the fill-in reaction.

The BEST protocol flanks the native input DNA molecules with adapters, which will include uracil bases. A uracil-tolerant polymerase must therefore be used during library amplification.

SsDNA2.0 (Figure 1B) is a single-stranded library preparation method optimized for damaged and degraded DNA and is the current state-of-the-art ssDNA method for highly degraded samples. We prepared ssDNA2.0 libraries as described in Gansauge et al. (Gansauge et al. 2017) using the TL136 splinter oligo (Supplemental Table 2).

Briefly, ssDNA2.0 libraries are prepared by first dephosphorylating the 5′ and 3′ termini of the input DNA with FastAP (Thermo Scientific, Waltham, MA). The DNA is then heat denatured at 95°C for 1 minute and then rapidly cooled in an ice bath. Once cooled, a biotinylated splinted adapter is ligated to the 3′ end of input DNA using T4 DNA Ligase (Thermo Scientific). The biotinylated adapters, including the ligation products, are then immobilized on C1 beads (Invitrogen), pulled down, and washed. An extension primer is then annealed to the ligated adapter and a second strand is synthesized using the Klenow Fragment (Thermo Scientific), followed by a second C1 bead pull-down and wash step. T4 DNA Ligase (Thermo Scientific) is then used to ligate a double-stranded blunt-end adapter to the 3′ end of the synthesized strand, followed by a third C1 bead pull-down and wash step. Finally, the reactions are heat denatured and the pre-amplified library is collected with the supernatant.

The final adapter-flanked product of an ssDNA2.0 library is the synthesized second strand. Because this will not contain uracil bases, a high fidelity and/or non-uracil tolerant polymerase can be used during library amplification. However, non-standard Illumina oligonucleotide design differences lead to a truncated P5 adapter, which requires the use of a non-standard Illumina sequencing primer.

The Santa Cruz Reaction (SCR; Figure 1C) uses directional splinted ligation of Illumina’s P5 and P7 adapters to convert natively single-stranded DNA and heat denatured double-stranded DNA into Illumina libraries in one enzymatic reaction. Similar to other library preparation protocols, including BEST and NEB Ultra II, the SCR scales the concentration of reaction components to the amount of input DNA to reduce the proportion of adapter-dimers. In the case of the SCR, that includes Extreme Thermostable Single-Stranded Binding Proteins (ET SSB, NEB), which scales with the amount of single-stranded DNA in the reaction. We recommend preparing several splinted adapter and ET SSB dilutions to be used for specific ranges of input DNA (see Supplemental Information).

The SCR begins by combining 20 µL of a DNA extract with 2 µL ET SSB (NEB) at a dilution optimized for the amount of input DNA (see Supplementary Information) to create a sample mixture. The sample mixture is then denatured by heating to 95°C for 3 min, followed by rapid cooling in an ice bath. Next, 1 µL each of P5 and P7 splinted adapters (also at dilutions optimized for the amount of input DNA; see Supplement) are added to the sample mix. Finally, 26 µL of SCR master mix containing 3.75 µL SCR Buffer (666 mm Tris–HCl, 132 mm MgCl₂), 0.5 µL 100 mm ATP (Thermo Scientific), 0.5 µL 1m DTT (Thermo Scientific), 0.625 µL 2 000 000 U/mL T4 DNA Ligase (NEB), 0.625 µL 10 000 U/mL T4 Polynucleotide Kinase (NEB), and 20 µL 50% PEG 8000 (NEB) is added to the sample mixture, creating a 50 µL reaction. The reaction is pulse-vortexed for 30 s, incubated at 37°C for 45 min, and then cleaned with a MinElute column following the manufacturer’s instructions.

Because the SCR ligates adapters directly to the native input molecules, a uracil tolerant polymerase must be used during library amplification.

The SCR is an ancient DNA-specific version of SRSLY, which was described by Troll et al. (Troll et al. 2019). Several alterations make the SCR more appropriate than SRSLY for converting damaged DNA. For example, the SCR uses DTT and ATP in place of T4 DNA Ligase Buffer (NEB), which appears to better stimulate T4 PNK. Because adapter-dimers are problematic when working with degraded and low-input samples, the SCR also recommends a series of the splinted adapter and ET SSB dilutions for lower DNA input volumes, and implements an asymmetric P5:P7 adapter molar ratio that reduces adapter-dimer formation. Finally, like ssDNA 2.0, the SCR adapter hybridization strategy uses a molar excess of splints to reduce the chance of splintless adapters in the reaction (see Supplementary Information).