TITLE: Non-UDG treated double-stranded DNA library preparation for Illumina sequencing of ancient dental calculus 
AUTHORS: Franziska Aron, Gunnar Neumann, Christina Warinner, 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). This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols.</div><div class = "text-block"><span>This protocol does </span><span style = "font-weight:bold;">not</span><span> include UDG (USER) treatment, in order to retain molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) 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></div></div>

[STEPS]
?. [Blunt End Repair (aDNA library preparation room)]
Prepare a mastermix for the blunt end repair calculating  . Use a new 1.5 ml LoBind tube to set up the mastermix.  ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2NEB Buffer 210 x1 x53ATP10 mM1 mM54BSA20 mg/ml0.8 mg/ml25dNTPs25 mM each0.1 mM0.26T4 PNK10 U0.4 U27T4 Polymerase3 U0.024 U0.48UV HPLC-water15.49DNA or UV HPLC-water2010Total50
[/ reaction]
ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2NEB Buffer 210 x1 x53ATP10 mM1 mM54BSA20 mg/ml0.8 mg/ml25dNTPs25 mM each0.1 mM0.26T4 PNK10 U0.4 U27T4 Polymerase3 U0.024 U0.48UV HPLC-water15.49DNA or UV HPLC-water2010Total50
You can use up to   extract, but with a maximum Input of  DNA in total.
?. [Blunt End Repair (aDNA library preparation room)]
Add  mastermix to each reaction tube and then add sample DNA to each tube (use 0.2 ml PCR tube strips). Mix by flicking the tubes and spin down briefly.
30 µl
20 µl
?. [Blunt End Repair (aDNA library preparation room)]
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 per sample and one set of 0.2 ml PCR tubes 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 3.2.
?. [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 (binding) buffer to a new 1.5 ml LoBind tube and add the blunt end repaired sample, 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 3.4.
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 in 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.
?. [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 3.10185Total40
[/ 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 3.10185Total40
?. [Adapter Ligation]
Aliquot  of mastermix to each tube (use 0.2 ml PCR strips). Transfer the complete eluate (step 3.10) (~) per sample to each new tube.
21 µl
18 µl
?. [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 7.2.
?. [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 3.4.
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 3.4.
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 in 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.
?. [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 7.10207Assay 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 7.10207Assay total40
?. [Adapter Fill-in]
Add of mastermix and the complete eluate (step 7.10) (~) 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 10 and new 1.5 ml LoBind tubes for step 11.
?. [Adapter Fill-in]
Aliquot  water to the 0.2 ml PCR tubes. Aliquot  per library to 0.2 ml PCR tubes with 18 uL water (making a 1:10 dilution of the library) for the qPCR quality check (steps 13-14).
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)]
Amplify the qPCR reactions 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 determined with this qPCR is used to determine the amount of sample used as input for indexing protocols.