A Fast and Efficient Single-stranded Genomic Library Preparation Method Optimized for Ancient DNA

Abstract We present a protocol to prepare extracted DNA for sequencing on the Illumina sequencing platform that has been optimized for ancient and degraded DNA. Our approach, the Santa Cruz Reaction or SCR, uses directional splinted ligation of Illumina’s P5 and P7 adapters to convert natively single-stranded DNA and heat denatured double-stranded DNA into sequencing libraries in a single enzymatic reaction. To demonstrate its efficacy in converting degraded DNA molecules, we prepare 5 ancient DNA extracts into sequencing libraries using the SCR and 2 of the most commonly used approaches for preparing degraded DNA for sequencing: BEST, which targets and converts double-stranded DNA, and ssDNA2.0, which targets and converts single-stranded DNA. We then compare the efficiency with which each approach recovers unique molecules, or library complexity, given a standard amount of DNA input. We find that the SCR consistently outperforms the BEST protocol in recovering unique molecules and, despite its relative simplicity to perform and low cost per library, has similar performance to ssDNA2.0 across a wide range of DNA inputs. The SCR is a cost- and time-efficient approach that minimizes the loss of unique molecules and makes accessible a taxonomically, geographically, and a temporally broader sample of preserved remains for genomic analysis.


S9. The Santa Cruz Reaction (SCR) Workflow
Note: The SCR uses a tiered adapter and SSB system based on the DNA input into the reaction. Use the table in Section 8 to select the correct dilution set for each DNA extract.
Critical Note: Thoroughly mixing the reaction in steps seven and eight is essential to consistently achieve high ligation efficiency. Inadequately mixing the reaction is the most common failure mode. Vortexing is recommended.
1. Gather the following reagents and equipment: • Thaw and equilibrate the Reaction Mix Preparation and appropriate SSB dilution to room temperature before pipetting. • Thaw the appropriate P5 and P7 adapter-splint dilutions and place them on ice.
• Prepare an ice bath. 11. The purified reaction products are ready for indexing and amplification by PCR.
12. A 1.2X SPRI purification is the recommended post-PCR purification strategy.

S10. Oligonucleotide Quality Control -Workflow Note:
The oligonucleotides for the Santa Cruz Reaction are designed with blocking modifications to limit undesirable ligation products. However, the splint oligonucleotides may arrive with one or more subspecies containing unblocked termini. Currently, a consistent purification strategy to eliminate poor quality splint species does not exist. However, this section presents a ligation based method to identify poor quality splint batches.
Briefly, splint oligonucleotides are spiked into Santa Cruz Reactions. Ligatable and amplifiable species within the splint spike will convert to library molecules, which will be identifiable during post amplification visualization. A splint batch with a high proportion of ligatable and amplifiable species should not be used for the library preparation of ancient samples (see section 11 for trace interpretation guidance).
Each new batch of P5 and P7 splint should undergo the quality control scheme below.
2. Prepare a number of Santa Cruz Reactions equal to the number of freshly synthesized splint batches requiring quality control plus one blank • Example: 1 P5 Splint + 1 P7 Splint + 1 Blank = 3 reactions.
3. In a 0.2 mL PCR 8-tube strip add the following to wells 1-3.

kapp_scr_suppMaterial_sub_v2
A fast and efficient single-stranded genomic library preparation method optimized for ancient DNA 5. Follow steps 3-5 of the SCR workflow in section eight to mix, heat denature, and chill the reactions.
8. Following incubation at 37 °C, clean each reaction using the MinElute PCR Purification Kit according to the manufacturer's instructions. Elute in 50 µL buffer EBT.
9. Amplify and index the entire eluate from step 8 for 18 cycles using the PCR scheme of your choice.
10. Clean each amplified library with the MinElute PCR Purification Kit according to the manufacturer's instructions.
11. Visualize each library, including the negative control, on a Fragment Analyzer, TapeStation, or BioAnalyzer automated electrophoresis system.

S12. Oligonucleotide Quality Control -Interpretation
Note: Clean splint batch libraries contain adapter-dimers and a small proportion of secondary oligonucleotide artifacts, which should resemble panel A and B in Figure S1. Note: Poorly synthesized splint batch libraries contain adapter-dimers and an unpredictable number of secondary oligonucleotide artifacts, which make-up a large portion of the library. Panel C and D in Figure S1 are examples or poorly synthesized splint batches, which should be discarded to avoid artifacts from contaminating the final libraries. Figure S1.

Compare the negative control trace to panels A and B in
• The adapter-dimer peak, around 130-140 bp, should dominate the trace but a small proportion of secondary peaks starting directly to the right of the adapter dimer and extending to 280bp may also be present. • The splint oligonucleotides are ready for use if the negative control is comparable to panels A and B in Figure S1. • See Step 2 if peaks to the right of the adapter-dimer peak dominate the negative control trace.

Compare the P5 and P7 splint input traces to the negative control trace.
• Between the P5 splint trace and negative control trace look for a change in the proportions of each peak. Do the same between the P7 splint trace and the negative control trace. • An observed decrease in the total proportion of the adapter-dimer peak (increase in secondary peaks) in one of the splint traces indicates the presence of undesirable oligonucleotide contaminants. • Quarantine or discard splint batches that have produced significant secondary oligonucleotide ligation artifacts.     A fast and efficient single-stranded genomic library preparation method optimized for ancient DNA Table S1. Overview of the ancient samples used for DNA extraction and library preparation. Table S2. Information for oligonucleotides used during library preparation. Table S3. Summary of library sequencing statistics.