Ambrecht et al. 2020: An optimized method for the extraction of ancient eukaryote DNA from marine sediments

Libraries containing 20 μl input DNA and small DNA fragments only (“Si_s”)

To target relatively small DNA fragments typical of aDNA, we per- formed a ‘reverse AxyPrep’ DNA purification post-library prepa- ration. For this, we prepared four additional libraries as described in Section 2.3.1.2, except that we added AxyPrep beads in a ratio 1:0.4 library:beads in the first clean-up step (aiming to retain frag- ments ≤500 bp). After 15 min of incubation and 15 min on a mag- netic rack to separate beads from the supernatant, we transferred the supernatant into a new sterile 1.7 ml tube. AxyPrep beads were added in a ratio 1:1.2 library:beads. Following two washes with 80% EtOH, we eluted the DNA in 30 μl Buffer EB with 0.05% Tween 20.

All libraries prepared in Sections 2.3.1.1–2.3.1.3 (23 in total) were quantified using the Qubit dsDNA HS Assay (Molecular Probes).

Based on the Qubit results, we designed a sequencing pool contain- ing 4 ng of DNA per library. As the EBCs contained only traces of DNA, we also prepared a second sequencing pool with 10 μl per EBC library. We then ran a PCR on these two sequencing pools (eight replicates with 25 μl reactions each) following Section 2.3.1, except that we used Indexing Primer IS4 and the GAII index 1 (Meyer & Kircher, 2010) and 13 cycles. Post-PCR quantifications of both pools were conducted using Qubit and merged into a third sequencing pool. To avoid over-diluting the latter sequencing pool, we used 4 ng DNA from pool 1 and 0.4 ng DNA from pool 2. The DNA quality and quantity of the third sequencing pool was assessed through TapeStation (Agilent Technologies). AxyPrep clean-up (at a ratio of 1:1.1 library:beads) and TapeStation checks were repeated twice to generate a high-quality sequencing pool (i.e., a minimal amount of primer-dimer, DNA concentration = 6.36 ng/μl). We submitted the final pool to the Australian Cancer Research Foundation Cancer Genomics Facility & Centre for Cancer Biology, Adelaide, Australia (hereafter ACRF) for Illumina NextSeq sequencing (2 × 75 bp cycle).

Libraries containing 20 μl input DNA (“Si20_20”)

In order to test whether relatively low or high amounts of input DNA affected library quality and sequencing results (particularly, whether PCR inhibition might become problematic, or whether larger DNA input volumes would benefit sequencing), we prepared four addi- tional libraries based on the DNA extracts derived from the Si20 ex- traction (three sediment extracts and one EBC; see Section 2.2.3). For these libraries we used 10× DNA input, totalling 20 μl per sam- ple, then followed Section 2.3.1.1 (Figure 1).

This technique was applied to 0.25g of sediment subsamples stored at 4°C, following the manufacturer's protocol with the some modifications:

TRANSFER sediment into individual bead-tubes using a disposable, sterile spatula

APPLY bead-beating in three runs of 0h 0m 20s with 0h 0m 5s breaks using a Precellys 24 homogenizer

CENTRIFUGE at 10319 for 0h 0m 30s

RETAIN all optional 0h 5m 0s incubation steps at 4°C per the kit's protocol

ELUTE DNA in 80µL of Buffer EB instead of the customary C6 solution and store at -20°C

This lysis process was applied to 0.25g of the subsamples stored at both 4°C and -20°C

FOLLOW the same protocol as described in Section 1 down to step 10 of the manufacturer's instructions (addition of Solution C3 and subsequent centrifugation)

AFTER this step...

TRANSFER the supernatant to 15 mL centrifuge tubes containing a DNA-binding buffer

STIR on a rotary mixer for at Room temperature

CENTRIFUGE at 4500rpm for 0h 5m 0s

DISCARD supernatant

RESUSPEND pellet in 900µL of DNA-binding buffer

RE-CENTRIFUGE at 14000rpm for 0h 1m 0s

DISCARD the supernatant

WASH the pellet twice in 80% ethanol

DRY pellet for 0h 15m 0s at 37°C

RESUSPEND pellet in 80µL Buffer EB

FOLLOWING incubation for 0h 10m 0s at 50°C, centrifuge at 14.000rpm,0h 0m 0s for 0h 1m 0s

STORE the supernatant (free of silica) in a sterile Lo-bind tube (Eppendorf) at -20°C

This technique was applied to 0.25g sediment subsamples stored at 4°C following Slon et al. (2017) with minor modifications

ADD 1mL of ethylenediaminetetraacetic acid (EDTA) to the sediment in a 2 mL screw-cap tube

PLACE samples on a rotary mixer and mix at 25rpm,0h 0m 0s, at Room temperature

CENTRIFUGE at 13.000rpm,0h 0m 0s for 0h 3m 0s

PURIFY the DNA using the MinElute Kit (Qiagen) as per the manufacturer's instructions

BIND DNA using the kit's spin column

ELUTE the DNA in 80µL of Buffer EB

INCUBATE 0.25g of three frozen sediment subsamples in EDTA overnight as in step 3.1 of the above section "EDTA + MinElute (“EDTA”)" , EXCEPT use only 0.75mL to keep volumes consistent with a subsequent step (below)

CENTRIFUGE at 13.000rpm,0h 0m 0s for 0h 3m 0s

KEEP supernatant at 4°C

PROCESS pellet separately using bead-beating and DNA purification, as in Method 2 (above)

RECOMBINE the resulting 0.75mL DNA-solution purified from the pellet (step 10 of DNeasy Kit protocol) with 0.75mL EDTA supernatant to make 1.5mL total

ADD 6mL modified QG buffer with 100µL liquid silica

PROCEED as described in Method 2 (above)

ELUTE the DNA in 100µL Buffer EB