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

[DESCRIPTION]

Four combinations of sedaDNA extraction treatments using marine sediments collected at a water depth of 104 m off Maria Island in Tasmania are compared. These methods contrast frozen versus refrigerated sediment, bead-beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica-solution.

All four methods worked to varying degrees; see paper for recommended shotgun library preparation


[BEFORE_START]
Sediment processing and pretreatment:
Core section processing, sedaDNA extractions and sequencing library preparations took place at ACAD's ultraclean forensic facilities following aDNA decontamination standards (Willerslev & Cooper, 2005). We placed the three sediment core sections into zip-lock bags sterilised with UV light and manually homogenized them for ~5 min. From each section, two 1 cm3 subsamples were transferred into two separate 15 ml centrifuge tubes using a sterile disposable spatula. One subsample was kept at 4°C and the other at –20°C for one month. The samples were prepared for the different extraction methods in a glove box decontaminated (3% bleach) between consecutive subsamples.

[GUIDELINES]
DNA extractions: 
Bead-beating + spin column (DNeasy PowerLyzer PowerSoil Kit, Qiagen; “Kit”)
Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)
EDTA + MinElute (“EDTA”) 
EDTA + bead-beating + liquid silica in QG Buffer (“Combined”, or “Com”) 

[STEPS]
SECTION: Method 1: Bead-beating + spin column (DNeasy PowerLyzer PowerSoil Kit, Qiagen; “Kit”)
1. This technique was applied to 0.25 g of sediment subsamples stored at 4 °C, following the manufacturer's protocol with the some modifications:
SECTION: Shotgun sequencing library preparation
None. We prepared four libraries as described in Section 2.3.1.2 down to the step of first post-PCR AxyPrep clean-up and Qubit quantification. Due to the low number of libraries, and to avoid over-diluting the final sequencing pool, we combined the four libraries to achieve a concen- tration of 4 ng/μl per sediment-derived library and a concentration of 0.4 ng μl for the EBC. We then ran GAII indexing PCR (Index 8), two TapeStation checks and one AxyPrep clean-up as in Section 2.3.1.1. Aiming to retain only the smallest DNA fragments (≤500 bp) from the full content of DNA detected by TapeStation, we added two rounds of the AxyBeads purification as in Section 2.3.1.3, with an initial library:beads ratio of 1:0.4 in round 1 and 1:0.6 in round 2 (Urich, Nery, Lister, Schmitz, & Ecker, 2015). As HMW DNA appeared to persist, we prepared a 1:10 dilution of the pool and repeated the AxyBeads size- selection one more time with an initial library:beads ratio of 1:0.6. We submitted the final pool (nondetectable primer-dimer, DNA concen- tration between 147 and 347 bp = 1.48 ng/μl) to ACRF for Illumina NextSeq sequencing (2 × 75 bp cycle). 
SECTION: Shotgun sequencing library preparation
0.30. 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). 



SECTION: Shotgun sequencing library preparation
0.30. 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). 



SECTION: Libraries from DNA extracts using the Kit, Si (Si4 and Si20), and EDTA techniques
None. Libraries containing 2 μl input DNA (“Si4”, “Si20”) 

We prepared libraries from all DNA extracts (i.e., a total of 15) after Weyrich et al. (2017) with the following modifications. A 2 μl aliquot of DNA was repaired (15 min, 25°C) in a 40 μl reaction using T4 DNA polymerase (New England Biolabs). After purifying the DNA (MinElute Reaction Cleanup Kit, Qiagen), a ligation step followed (T4 DNA ligase, Fermentas) in which truncated Illumina-adapter sequences containing two unique five base-pair (bp) barcodes were attached to the double- stranded DNA (60 min, 22°C; Meyer & Kircher, 2010). An additional DNA purification (MinElute Reaction Cleanup Kit, Qiagen) step was performed, followed by a fill-in reaction with adapter sequences (Bst DNA polymerase, New England Biolabs; 30 min, 37°C, with polymer- ase deactivation for 10 min, 80°C). We then used 3 μl of the reaction- product as input for a 25 μl PCR (eight replicates per extract) with the primers IS7 and IS8 (Meyer & Kircher, 2010). Each PCR reaction in- cluded 14.2 nuclease-free H2O, 2.5 μl 10× Gold Buffer, 2.5 μl 25 mM MgCl2, 0.25 μl 25 mM dNTPs, 1.25 IS7, 1.25 IS8, and 0.1 μl AmpliTaq Gold Polymerase (Applied Biosystems). Thermal cycling specifications were as follows: 6 min at 94°C, 13 cycles of 30 s denaturation at 94°C, 30 s annealing at 60°C, 40 s extension at 72°C, and 10 min of final extension. We purified PCR products using AxyPrep magnetic beads (Axygen Biosciences; 1:1.8 library:beads) and eluted the DNA in Buffer EB (Qiagen) with 0.05% Tween 20 (Sigma Aldrich). 

SECTION: Method 4: EDTA + bead-beating + liquid silica in QG Buffer (“Combined”, or “Com”)
4. INCUBATE 0.25 g of three frozen sediment subsamples in EDTA overnight as in step 3.1 of the above section "EDTA + MinElute (“EDTA”)", EXCEPT use only 0.75 mL to keep volumes consistent with a subsequent step (below)
SECTION: Method 3: EDTA + MinElute (“EDTA”)
3.3. BIND DNA using the kit's spin column

ELUTE the DNA in 80 µL of Buffer EB
SECTION: Method 3: EDTA + MinElute (“EDTA”)
3.2. CENTRIFUGE at 13.000 rpm for 3 min

PURIFY the DNA using the MinElute Kit (Qiagen) as per the manufacturer's instructions
SECTION: Method 3: EDTA + MinElute (“EDTA”)
3.1. ADD 1 mL of ethylenediaminetetraacetic acid (EDTA) to the sediment in a 2 mL screw-cap tube 

PLACE samples on a rotary mixer and mix at 25 rpm,   at Room temperature
SECTION: Method 3: EDTA + MinElute (“EDTA”)
3. This technique was applied to 0.25 g sediment subsamples stored at 4 °C following Slon et al. (2017) with minor modifications
SECTION: Method 2: Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)
2.5. FOLLOWING incubation for 10 min at 50 °C, centrifuge at 14.000 rpm for 1 min  

STORE the supernatant (free of silica) in a sterile Lo-bind tube (Eppendorf) at -20 °C
SECTION: Method 2: Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)
2.4. RE-CENTRIFUGE at 14000 rpm for 1 min

DISCARD the supernatant

WASH the pellet twice in 80 % ethanol

DRY pellet for 15 min at 37 °C 

RESUSPEND pellet in 80 µL Buffer EB
SECTION: Method 2: Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)
2.3. STIR on a rotary mixer for   at Room temperature 

CENTRIFUGE at 4500 rpm for 5 min 

DISCARD supernatant

RESUSPEND pellet in 900 µL of DNA-binding buffer
SECTION: Method 2: Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)
2.2. TRANSFER the supernatant to 15 mL centrifuge tubes containing a DNA-binding buffer
 
 
SECTION: Method 2: Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)
2.1. 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...
SECTION: Method 2: Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)
2. This lysis process was applied to 0.25 g of the subsamples stored at both 4 °C and -20 °C
SECTION: Method 1: Bead-beating + spin column (DNeasy PowerLyzer PowerSoil Kit, Qiagen; “Kit”)
1.4. ELUTE DNA in 80 µL of Buffer EB instead of the customary C6 solution and store at -20 °C
SECTION: Method 1: Bead-beating + spin column (DNeasy PowerLyzer PowerSoil Kit, Qiagen; “Kit”)
1.3. RETAIN all optional 5 min incubation steps at 4 °C per the kit's protocol
SECTION: Method 1: Bead-beating + spin column (DNeasy PowerLyzer PowerSoil Kit, Qiagen; “Kit”)
1.2. APPLY bead-beating in three runs of 20 s with 5 s breaks using a Precellys 24 homogenizer

CENTRIFUGE at 10319 2496  for 30 s 
SECTION: Method 1: Bead-beating + spin column (DNeasy PowerLyzer PowerSoil Kit, Qiagen; “Kit”)
1.1. TRANSFER  sediment into individual bead-tubes using a disposable, sterile spatula
SECTION: Libraries from DNA extracts using the Kit, Si (Si4 and Si20), and EDTA techniques
None. 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). 
SECTION: Libraries from DNA extracts using the Kit, Si (Si4 and Si20), and EDTA techniques
None. 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). 
SECTION: Libraries from DNA extracts using the Kit, Si (Si4 and Si20), and EDTA techniques
None. 
SECTION: Libraries from DNA extracts using the Kit, Si (Si4 and Si20), and EDTA techniques
None. 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, we prepared four additional libraries based on the DNA extracts derived from the Si20 extraction (see Section "Bead-beating + liquid silica in QG Buffer (“Si4” and “Si20”)"). For these libraries we used 10× DNA input, totaling 20 µL per sample, then followed Section 2.3.1.1 (Figure 1). 
SECTION: Libraries from DNA extracts using the Kit, Si (Si4 and Si20), and EDTA techniques
None. Libraries containing 20 μl input DNA and small DNA fragments only (“Si_s”)
To target relatively small DNA fragments typical of aDNA, we performed a ‘reverse AxyPrep’ DNA purification post-library preparation. 
SECTION: Method 4: EDTA + bead-beating + liquid silica in QG Buffer (“Combined”, or “Com”)
4.1. CENTRIFUGE at 13.000 rpm for 3 min  

KEEP supernatant at 4 °C

PROCESS pellet separately using bead-beating and DNA purification, as in Method 2 (above)
SECTION: Method 4: EDTA + bead-beating + liquid silica in QG Buffer (“Combined”, or “Com”)
4.2. RECOMBINE the resulting 0.75 mL DNA-solution purified from the pellet (step 10 of DNeasy Kit protocol) with 0.75 mL EDTA supernatant to make 1.5 mL total

ADD 6 mL modified QG buffer with 100 µL liquid silica

PROCEED as described in Method 2 (above)
SECTION: Method 4: EDTA + bead-beating + liquid silica in QG Buffer (“Combined”, or “Com”)
4.3. ELUTE the DNA in 100 µL Buffer EB