TITLE: Half-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing
AUTHORS: Franziska Aron, Gunnar Neumann, Guido Brandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA) with partial USER enzyme treatment. This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols.</div><div class = "text-block">The partial uracil DNA glycosylase (UDG) (USER enzyme) treatment reduces molecular 'damage' in the form of deaminated cytosines characteristic of aDNA in the interior of the DNA molecules but leaves this damage on both the 5' and 3' ends. </div><div class = "text-block">This protocol is modified after: </div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Rohland et al. (2015): Partial uracil–DNA–glycosylase treatment for screening of ancient DNA. Phil. Trans. R. Soc. B370: 20130624 (</span><a href="http://dx.doi.org/10.1098/rstb.2013.0624" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">doi.org/10.1098/rstb.2013.0624</span></a><span>) </span></li><li style = "counter-reset:ol0;list-style-type:disc;">and </li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Meyer & Kircher (2010): Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing. Cold Spring Harb. Protoc. (doi: </span><a href="https://dx.doi.org/10.1101/pdb.prot5448" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1101/pdb.prot5448</span></a><span>).</span></li></ul></div><div class = "text-block">and </div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. [UDG Treatment (aDNA library preparation room)]
Prepare a mastermix for the UDG (USER enzyme) treatment calculating  . Use a new 1.5 ml LoBind tube to set up the mastermix.  ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2Buffer Tango10 x1.2 x63ATP10 mM1.2 mM64BSA20 mg/ml0.2 mg/ml0.55dNTPs25 mM each0.1 mM each0.26USER Enyzme1 U0.072 U3.67DNA or UV HPLC-Water258UV HPLC-Water8.709Total50
[/ reaction]
ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2Buffer Tango10 x1.2 x63ATP10 mM1.2 mM64BSA20 mg/ml0.2 mg/ml0.55dNTPs25 mM each0.1 mM each0.26USER Enyzme1 U0.072 U3.67DNA or UV HPLC-Water258UV HPLC-Water8.709Total50
?. [UDG Treatment (aDNA library preparation room)]
Add  mastermix to each reaction tube (0.2 ml PCR tube) and then add sample DNA extract to each tube. Mix by flicking the tubes then spin down briefly.
25 µl
25 µl
?. [UDG Treatment (aDNA library preparation room)]
Incubate at  for , then at  for  in the thermocycler.
37 °C
12 °C
?. [MinElute Purification]
Purify the blunt-end repaired DNA with a MinElute kit, with the following modifications to the manufacturer's protocol.
?. [MinElute Purification]
Pre-heat elution buffer EBT to .
50 °C
?. [MinElute Purification]
For each reaction, add  PB buffer to a new 1.5 ml LoBind tube and add the blunt-end repaired sample from step 6, then vortex briefly to mix.
650 µl
?. [MinElute Purification]
Load each reaction (PB buffer + blunt-end repaired sample) onto a MinElute column and incubate at RT for .
This allows sufficient time for the DNA to bind to the silica membrane.
?. [MinElute Purification]
Spin    and discard flow-through.
Centrifuge: 15800 34, 1 min
Discard flow-through in one of two following ways: Remove all liquid in the collection tube with a pipette, orPour off the liquid into a fresh waste tube, and pat the rim of the collection tube dry on a paper tissue or towel. Use just one spot on the paper tissue per sample. Be careful not to touch the rim of the tube on the waste container. After you are finished with all samples, discard the paper and wipe clean the surface underneath with water and then sterilize the surface with bleach (or DNA Away).
During cleaning of surfaces after flow-through discard, do NOT apply bleach before first cleaning with water. Residual GuHCl will react with bleach to form toxic gases.
?. [MinElute Purification]
Add  PE (wash) buffer to the MinElute column.
700 µl
?. [MinElute Purification]
Spin at    and discard flow-through as in step 7.4.
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Dry spin for  .
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Flip columns 180° and dry spin again for  .
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Remove columns from their collection tubes and place them into new 1.5 ml LoBind tubes.
?. [MinElute Purification]
Add    pre-heated EBT to the column, let stand for  then spin  to elute.
20 µl
Centrifuge: 15800 34, 1 min
Carefully pipette EBT directly onto the center of the membrane without touching the membrane.Optional Freezing Point for up to two weeks at
?. [Adapter Ligation]
Prepare a mastermix for adapter ligation calculating . Use a 1.5 ml LoBind tube to set up the ligation mastermix.  ABCD1ReagentStock concentrationFinal concentration1 X Volume [µl]2Quick Ligase Buffer2 x1 x203Adapter Mix10 µM0.25 µM14Eluate from Step 7.10185Total40
[/ reaction]
The mastermix is calculated to accommodate addition of Quick Ligase to each reaction individually after aliquotting to individual reaction tubes.
ABCD1ReagentStock concentrationFinal concentration1 X Volume [µl]2Quick Ligase Buffer2 x1 x203Adapter Mix10 µM0.25 µM14Eluate from Step 7.10185Total40
?. [Adapter Ligation]
Aliquot  of mastermix to each tube (use 0.2 ml PCR strips). Transfer the complete eluate (step 7.10) (~) per sample to each new tube.
21 µl
18 µl
Alternatively, if you have a heat block available, you can add the mastermix to the eluate directly in the 1.5 ml tubes from the elution step without transferring it into 0.2 ml PCR strips. For purification after ligation you can then also add the PB-Buffer into these tubes (step 11.2).
?. [Adapter Ligation]
Then add  of Quick ligase ( stock,  final concentration) to each library sample. Mix by flicking the tubes and spin down.
1 µl
Make sure to add the full volume of ligase by pipetting up and down several times to get all of it off the walls of the pipette tip.
?. [Adapter Ligation]
Incubate at for  in the thermocycler.
22 °C
During this incubation take MinElute tubes out of the fridge so that they warm to room temperature before use in the next step. Also label two sets of 1.5 ml LoBind tubes and 0.2 ml PCR tubes per sample for the next steps. At this time you can also add the PB-Buffer (650µl) to one set of 1.5 ml LoBind tubes for Step 11.2.
?. [MinElute Purification]
Purify the adapter-ligated libraries with a MinElute kit, with the following modifications to the manufacturer's instructions
?. [MinElute Purification]
Pre-heat EBT to  .
50 °C
?. [MinElute Purification]
For each sample, add  PB buffer to a new 1.5 ml LoBind tube and add the entire volume of the adapter ligation mix, then vortex briefly.
650 µl
?. [MinElute Purification]
Load each reaction (PB buffer + adapter-ligated library) onto a MinElute column and incubate for .
This allows the DNA sufficient time to bind to the silica membrane
?. [MinElute Purification]
Spin at   and discard flow-through as in step 7.4.
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Add  PE buffer to the MinElute column.
700 µl
?. [MinElute Purification]
Spin at   and discard flow-through as in step 7.4.
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Dry spin at  .
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Flip columns by 180° and dry spin again at   .
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Remove columns from their collection tubes and place them into new 1.5 ml LoBind tubes.
?. [MinElute Purification]
Add  of pre-heated EBT to column, let stand for , then spin  to elute.
22 µl
Centrifuge: 15800 34, 1 min
Carefully pipette EBT directly onto the center of the membrane without touching it.Optional Freezing Point for up to  two weeks at
?. [Adapter Fill-in]
Prepare adapter fill-in reaction calculating  . Use a 1.5 ml LoBind tube to set up the adapter fill-in mastermix.  ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2Isothermal Buffer10 x1 x43dNTPs25 mM each0.125 mM each0.24Bst Polymerase8 U0.4 U25UV HPLC-water13.86Eluate from step 11.10207Assay total40
[/ reaction]
ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2Isothermal Buffer10 x1 x43dNTPs25 mM each0.125 mM each0.24Bst Polymerase8 U0.4 U25UV HPLC-water13.86Eluate from step 11.10207Assay total40
?. [Adapter Fill-in]
Add of mastermix and the complete eluate (step 11.10) (~) to each tube (use 0.2 ml PCR strips). Mix by flicking the tubes and spin down briefly.
20 µl
20 µl
?. [Adapter Fill-in]
Incubate at  for  then  for  in the thermocycler.
37 °C
80 °C
During this incubation label new 0.2 ml tubes for step 14 and new 1.5 ml LoBind tubes for step 15.
?. [Adapter Fill-in]
Aliquot  water to the 0.2 ml PCR tubes. Aliquot  per library to 0.2 ml PCR tubes with 18 µl water (making a 1:10 dilution of the library) for the qPCR quality check (steps 17-18).
18 µl
2 µl
?. [Adapter Fill-in]
Transfer the remaining ~ of final library to a fresh 1.5 ml LoBind tube.
36 µl
?. [Adapter Fill-in]
Freeze the library at  without purification until further processing.
-20 °C
?. [qPCR_quality check (modern DNA facility)]
Prepare a qPCR assay calculating . Prepare 2 reactions per sample, plus 16 additional reactions for 7 qPCR standards in duplicates and 2 qPCR blanks. This qPCR uses a 1:10 dilution of the samples.   ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2DyNAmo MasterMix2 x1 x103IS7 primer10 µM1 µM14IS8 primer10 µM1 µM15HPLC-Water (non UVed)76DNA or HPLC-Water (1:10 dilution)17Total20
[/ reaction]
The 1:10 dilutions of the samples are prepared in the cleanroom, but the qPCR is performed in the modern lab.
ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2DyNAmo MasterMix2 x1 x103IS7 primer10 µM1 µM14IS8 primer10 µM1 µM15HPLC-Water (non UVed)76DNA or HPLC-Water (1:10 dilution)17Total20
**Important** Do NOT vortex the DyNAmo MasterMix, it will create bubbles that are nearly impossible to remove. The bubbles will interfere with the qPCR measurements. Gently pipette the DyNAmo MasterMix to equally distribute the fluorescent dye. Gently pipette the qPCR master mix, or gently invert the tube several times to mix. Again avoid  creating any bubbles.
?. [qPCR_quality check (modern DNA facility)]
Add mastermix and  1:10 diluted libraries, standard, or water to a fresh 96-well qPCR plate. Seal with adhesive clear qPCR seal sheets and briefly spin down.
19 µl
1 µl
Be sure to check that the reactions do not contain bubbles, because this will affect the qPCR readings and subsequent downstream calculations. If bubbles are present, briefly centrifuge the plate at maximum speed to remove them.
?. [qPCR_quality check (modern DNA facility)]
Run the qPCR assay with the following program:  ABC1TemperatureTime295°C10 minInital denaturation395°C30 sec40 cycles460°C1 min572°C30 sec660-95˚CMelting curve7Finally hold the reactions at 37°C.
ABC1TemperatureTime295°C10 minInital denaturation395°C30 sec40 cycles460°C1 min572°C30 sec660-95˚CMelting curve7Finally hold the reactions at 37°C.
The number of DNA copies calculated from this qPCR is used to determine the amount of sample used as input for indexing protocol.
?. [UDG inhibition]
Add  UGI (2 U Stock concentration, 0.1343 U final concentration) to each reaction.Mix by flicking the tubes and spin down briefly.
3.6 µl
?. [UDG inhibition]
Incubate at  for , then at  for  in the thermocycler.
37 °C
12 °C
?. [Blunt End Repair]
Prepare a mastermix for blunt-end repair of the DNA fragments, calculating  . Use a new 1.5ml LoBind tube to set up the master mix.   ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2T4 PNK10 U0.515 U33T4 Polymerase3 U0.085 U1.654Reaction from step 453.605Total58.25
[/reaction]
ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2T4 PNK10 U0.515 U33T4 Polymerase3 U0.085 U1.654Reaction from step 453.605Total58.25
?. [Blunt End Repair]
Incubate at  for , then at  for  in the thermocycler.
25 °C
12 °C
During this incubation take MinElute tubes out of the fridge so that they warm to room temperature before use in the next step. Also label two sets of 1.5 ml LoBind tubes and 0.2 ml PCR tubes per sample for the next steps. At this time you can also add the PB-Buffer (650µl) to one set of 1.5 ml LoBind tubes for Step 7.2.
?. [Blunt End Repair]
Add   mastermix (T4 PNK + T4 polymerase) to each reaction from step 4.Mix by flicking the tubes and spin down briefly
4.65 µl