TITLE: Illumina double-stranded DNA dual indexing for ancient DNA
AUTHORS: Raphaela Stahl, Christina Warinner, Irina Velsko, Eleftheria Orfanou, Franziska Aron, Guido Brandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol converts partially completed double-stranded DNA libraries e.g. from:</div><div class = "text-block">Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (dx.doi.org/10.17504/protocols.io.bakricv6)</div><div class = "text-block">into dual-indexed Illumina libraries ready for sequencing on an Illumina platform. Other variants of the above protocol can also be used.</div><div class = "text-block">This protocol includes steps for indexing PCR, clean-up, and post-indexing qPCR. This protocol is modified after Kircher, M., Sawyer, S. & Meyer, M., 2012. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic acids research, 40(1), p.e3. http://dx.doi.org/10.1093/nar/gkr771.                  </div></div>

[STEPS]
?. [Indexing preparation (aDNA library preparation room)]
Calculate the total number of DNA molecules (total copy number) DNA concentration in each library based on qPCR performed at the end of library preparation (see Before Start). Do not use more than 1.5x10^10 copies per indexing reaction. Adjust the amount of DNA used per reaction based on the initial library quantification. Depending on the total amount of DNA, split the indexing PCR into 2, 4, or 6 reactions per library.Store all remaining libraries eluates in the freezer at  for short-term storage (1-2 months) or the  freezer for long-term storage.
The number of indexing reactions per library depends on the DNA concentration of the library, which comes from the qPCR run at the end of library preparation (see Before Start).
If the total copy number is high enough that six splits are not sufficient to bring the number of DNA copies per reaction to 1.5x10^10, then calculate the amount of splits nessesary to stay below this threshold. Divide the total volume of the library ( ) by the number of splits to know the maximum input volume of library per index reaction. Set up a maximum of six indexing reactions. Keep the remaining DNA-Library as a backup at  .
-20 °C
-80 °C
?. [Indexing preparation (aDNA library preparation room)]
Assign unique dual index combinations of index primer pairs to each library.
?. [Splits: 2 (aDNA library preparation room)]
Calculate the amount of master mix needed for the number of samples being processed. Prepare the master mix according to the table below within a 1.5 mL LoBind tube. Add  mastermix,  of each index primer and sample to each tube (use 0.2 mL PCR strips).  ABCD1ReagentStock ConcentrationFinal concentration1× Volume [µl]2Pfu Turbo Cx Buffer10 ×1 ×10.03BSA20 mg/mL0.3 mg/mL1.54dNTPs25 mM each0.25 mM each1.05Pfu Turbo Cx Polymerase2.5 U0.025 U1.06Index P5_Jen_8nt10 µM0.22.07Index P7_Jen_8nt10 µM0.2 µM2.08UV HPLC-water--64.59DNA or UV HPLC-water--18.010Total100.0
78 µl
2 µl
18 µl
ABCD1ReagentStock ConcentrationFinal concentration1× Volume [µl]2Pfu Turbo Cx Buffer10 ×1 ×10.03BSA20 mg/mL0.3 mg/mL1.54dNTPs25 mM each0.25 mM each1.05Pfu Turbo Cx Polymerase2.5 U0.025 U1.06Index P5_Jen_8nt10 µM0.22.07Index P7_Jen_8nt10 µM0.2 µM2.08UV HPLC-water--64.59DNA or UV HPLC-water--18.010Total100.0
Each library will have 2 reactions of   each.
?. [Splits: 4 (aDNA library preparation room)]
Calculate the amount of master mix needed for the number of samples being processed. Prepare the master mix according to the table below within a 1.5 mL LoBind tube. Add  mastermix, of each index primer and  sample to each tube (use 0.2 mL PCR strips).  ABCD1ReagentStock conconcentrationFinal concentration1× Volume [µL]2Pfu Turbo Cx Buffer10 ×1 ×10.03BSA20 mg/mL0.3 mg/mL1.54dNTPs25 mM0.25 mM1.05Pfu Turbo Cx Polymerase2.5 U0.025 U1.06Index P5_Jen_8nt10 µM0.2 µM2.07Index P7_Jen_8nt10 µM0.2 µM2.08UV HPLC-water--73.59DNA or UV HPLC-water--9.010Total100.0
87 µl
2 µl
9 µl
ABCD1ReagentStock conconcentrationFinal concentration1× Volume [µL]2Pfu Turbo Cx Buffer10 ×1 ×10.03BSA20 mg/mL0.3 mg/mL1.54dNTPs25 mM0.25 mM1.05Pfu Turbo Cx Polymerase2.5 U0.025 U1.06Index P5_Jen_8nt10 µM0.2 µM2.07Index P7_Jen_8nt10 µM0.2 µM2.08UV HPLC-water--73.59DNA or UV HPLC-water--9.010Total100.0
Each library will have 4 reactions of    each.
?. [Splits: 6 (aDNA library preparation room)]
Calculate the amount of master mix needed for the number of samples being processed. Mix the master mix from the table below in a 1.5 mL LoBind tube. Add mastermix,  of each index primer and  sample to each tube (use 0.2 ml PCR strips).  ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2Pfu Turbo Cx Buffer10 ×1 ×10.03BSA20 mg/mL0.3 mg/mL1.54dNTPs25 mM each0.25 mM each1.05Pfu Turbo Cx Polymerase2.5 U0.025 U1.06Index P5_Jen_8nt10 µM0.2 µM2.07Index P7_Jen_8nt10 µM0.2 µM2.08UV HPLC-water--76.59DNA or UV HPLC-water--6.010Total100.0
90 µl
2 µl
6 µl
ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2Pfu Turbo Cx Buffer10 ×1 ×10.03BSA20 mg/mL0.3 mg/mL1.54dNTPs25 mM each0.25 mM each1.05Pfu Turbo Cx Polymerase2.5 U0.025 U1.06Index P5_Jen_8nt10 µM0.2 µM2.07Index P7_Jen_8nt10 µM0.2 µM2.08UV HPLC-water--76.59DNA or UV HPLC-water--6.010Total100.0
Each library will have 6 reactions of    each.
?. [Move to modern DNA lab]
Securely close the reactions and transfer to modern DNA laboratory. If possible, keep the reactions on ice during the transfer.
?. [Indexed adapter ligation with PCR (modern DNA lab)]
In a modern DNA lab, use a thermocycler to amplify the reactions with the following program:  ABC1TemperatureTime295°C2 minInital denaturation395°C30 sec10 cycles458°C30 sec572°C1 min672˚C10 minFinal elongation710˚Cuntil further processing
ABC1TemperatureTime295°C2 minInital denaturation395°C30 sec10 cycles458°C30 sec572°C1 min672˚C10 minFinal elongation710˚Cuntil further processing
During this incubation, label new 1.5mL LoBind tubes for post-purification elution (step 10).
?. [MinElute Purification (modern DNA lab)]
Purify the indexed libraries with a MinElute kit, with the following modifications to the manufacturer's protocol.
?. [MinElute Purification (modern DNA lab)]
For each reaction, add   PB (binding) buffer to a new 5 mL LoBind tube. Add the library, then vortex briefly to mix. A single column can be used for up to 4 index reactions of one library. Therefore, each PB and library mix of a single library that was split in 2 to 4 reactions will be loaded onto the same column.
650 µl
If 6 or more splits were performed for the indexing reactions,  the PB and library mix can be split over 2 columns for purification. In this case, elute the DNA from each column in step 8.8 with    EBT and combine the eluate to get  .
?. [MinElute Purification (modern DNA lab)]
Load each reaction (PB buffer + library) onto a MinElute column and incubate at RT for  .
This allows sufficient time for the DNA to bind to the silica membrane.
?. [MinElute Purification (modern DNA lab)]
Spin at   and discard flow-through.
Centrifuge: 15800 34, 1 min
Pour off the liquid into a 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 soap.
?. [MinElute Purification (modern DNA lab)]
Add   PE (wash) buffer to the MinElute column.
700 µl
?. [MinElute Purification (modern DNA lab)]
Spin at  and discard flow-through.
Centrifuge: 15800 34, 1 min
?. [MinElute Purification (modern DNA lab)]
Dry spin at  .
Centrifuge: 15800 34, 1 min
?. [MinElute Purification (modern DNA lab)]
Remove columns from their collection tubes and place them in new 1.5 mL LoBind tubes.
?. [MinElute Purification (modern DNA lab)]
Add  EBT to the column of the filter, let stand for , then spin at  to elute.
50 µl
Centrifuge: 15800 34, 1 min
Carefully pipette EBT directly onto the center of the membrane without touching the membrane.
If two columns were used for the purification of one library, elute each in  and pool both reactions to produce a total elution volume of  .
?. [qPCR Quality Check (modern DNA facility)]
Dilute  of the indexed library 1:1000 for qPCR. Do this in 2 steps: Make a 1:10 dilution, and then make a 1:100 dilution of the 1:10 dilution, for a final dilution of 1:1000.
2 µl
?. [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.  ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2DyNAmo Master Mix2 ×1 ×103IS5 primer10 µM0.5 µM14IS6 primer10 µM0.5 µM15HPLC-Water (non UVed)--76DNA or HPLC-Water (1:1000 dilution)--17Total20
[/reaction]
ABCD1ReagentStock concentrationFinal concentration1× Volume [µl]2DyNAmo Master Mix2 ×1 ×103IS5 primer10 µM0.5 µM14IS6 primer10 µM0.5 µM15HPLC-Water (non UVed)--76DNA or HPLC-Water (1:1000 dilution)--17Total20
Important Do NOT vortex the DyNAmo MasterMix, it will create bubbles that are very difficult 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 vortexing so you do not create any bubbles.
?. [qPCR Quality Check (modern DNA facility)]
Add  mastermix and  diluted libraries, standard, or water for each reaction to a fresh 96-well plate.
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 final amplification and pooling for sequencing (separate protocol).