Illumina double-stranded DNA dual indexing for ancient DNA

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 -20°C for short-term storage (1-2 months) or the -80°C freezer for long-term storage.

Assign unique dual index combinations of index primer pairs to each library.

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 78µL mastermix, 2µL of each index primer and 18µLsample to each tube (use 0.2 mL PCR strips).

A	B	C	D
Reagent	Stock Concentration	Final concentration	1× Volume [µl]
Pfu Turbo Cx Buffer	10 ×	1 ×	10.0
BSA	20 mg/mL	0.3 mg/mL	1.5
dNTPs	25 mM each	0.25 mM each	1.0
Pfu Turbo Cx Polymerase	2.5 U	0.025 U	1.0
Index P5_Jen_8nt	10
µM	0.2	2.0
Index P7_Jen_8nt	10 µM	0.2 µM	2.0
UV HPLC-water	-	-	64.5
DNA or UV HPLC-water	-	-	18.0
Total			100.0

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 87µL mastermix, 2µLof each index primer and 9µL sample to each tube (use 0.2 mL PCR strips).

A	B	C	D
Reagent	Stock conconcentration	Final concentration	1× Volume [µL]
Pfu Turbo Cx Buffer	10 ×	1 ×	10.0
BSA	20 mg/mL	0.3 mg/mL	1.5
dNTPs	25 mM	0.25 mM	1.0
Pfu Turbo Cx Polymerase	2.5 U	0.025 U	1.0
Index P5_Jen_8nt	10 µM	0.2 µM	2.0
Index P7_Jen_8nt	10 µM	0.2 µM	2.0
UV HPLC-water	-	-	73.5
DNA or UV HPLC-water	-	-	9.0
Total			100.0

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 90µLmastermix, 2µL of each index primer and 6µL sample to each tube (use 0.2 ml PCR strips).

A	B	C	D
Reagent	Stock concentration	Final concentration	1× Volume [µl]
Pfu Turbo Cx Buffer	10 ×	1 ×	10.0
BSA	20 mg/mL	0.3 mg/mL	1.5
dNTPs	25 mM each	0.25 mM each	1.0
Pfu Turbo Cx Polymerase	2.5 U	0.025 U	1.0
Index P5_Jen_8nt	10 µM	0.2 µM	2.0
Index P7_Jen_8nt	10 µM	0.2 µM	2.0
UV HPLC-water	-	-	76.5
DNA or UV HPLC-water	-	-	6.0
Total			100.0

Securely close the reactions and transfer to modern DNA laboratory. If possible, keep the reactions on ice during the transfer.

In a modern DNA lab, use a thermocycler to amplify the reactions with the following program:

A	B	C
Temperature	Time
95°C	2 min	Inital denaturation
95°C	30 sec	10 cycles
58°C	30 sec
72°C	1 min
72˚C	10 min	Final elongation
10˚C	until further processing

Purify the indexed libraries with a MinElute kit, with the following modifications to the manufacturer's protocol.

For each reaction, add 650µL 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.

Load each reaction (PB buffer + library) onto a MinElute column and incubate at RT for 0h 2m 0s .

Spin at 15800x g and discard flow-through.

Add 700µL PE (wash) buffer to the MinElute column.

Spin at 15800x g and discard flow-through.

Dry spin at 15800x g .

Remove columns from their collection tubes and place them in new 1.5 mL LoBind tubes.

Add 50µL EBT to the column of the filter, let stand for 0h 1m 0s, then spin at 15800x g to elute.

Dilute 2µL 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.

Prepare a qPCR assay calculating 20µL. Prepare 2 reactions per sample, plus 16 additional reactions for 7 qPCR standards in duplicates and 2 qPCR blanks.

A	B	C	D
Reagent	Stock concentration	Final concentration	1× Volume [µl]
DyNAmo Master Mix	2 ×	1 ×	10
IS5 primer	10 µM	0.5 µM	1
IS6 primer	10 µM	0.5 µM	1
HPLC-Water (non UVed)	-	-	7
DNA or HPLC-Water (1:1000 dilution)	-	-	1
Total			20

Do not add the DNA dilutions to the mastermix.

Add 19µL mastermix and 1µL diluted libraries, standard, or water for each reaction to a fresh 96-well plate.

Amplify the qPCR reactions with the following program:

A	B	C
Temperature	Time
95°C	10 min	Inital denaturation
95°C	30 sec	40 cycles
60°C	1 min
72°C	30 sec
60-95˚C		Melting curve
Finally hold the reactions at 37°C.