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

[DESCRIPTION]
This protocol converts partially completed double-stranded DNA libraries e.g. from:

Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (dx.doi.org/10.17504/protocols.io.bakricv6)

into dual-indexed Illumina libraries ready for sequencing on an Illumina platform. Other variants of the above protocol can also be used.

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.         

[BEFORE_START]
Planning
This protocol takes 1 day.

During the protocol samples move from the ancient DNA laboratory to a modern molecular biology lab: 
Indexing reactions are prepared in the ancient DNA lab, and then closed tubes are transferred to a standard modern molecular biology lab. All PCR amplification must take place in the modern molecular biology lab and NOT in the ancient DNA lab. 
 

Check waste disposal guidance for all reagents in this protocol against your corresponding laboratory regulations.

Preparation of buffers (Qiagen MinElute kit):
Add ethanol to PE wash buffer acccording to manufacturer's instructions.
Add Tween-20 to EB elution buffer to a final concentration of 0.05% (Tween-20 in EB). This solution is referred to as EBT throughout the protocol. 

Equipment
Make sure all necessary equipment is available (see Materials).

Abbreviations
EBT = modified EB-Buffer (MinElute Kit), see Preparation of buffers
HPLC = High Performance Liquid Chromatography (-Grade Water)
PE = PE-Buffer from Qiagen MinElute Kit
PB = PB-Buffer from Qiagen MinElute Kit
UV = Ultraviolet (radiation)

Samples
This protocol uses around 36 µL of the remaining eluate after the final qPCR from library preprartion (e.g. dx.doi.org/10.17504/protocols.io.bakricv6), that is stored at -20 °C in the ancient DNA lab.

Controls
Take along a positive control and 2 negative controls (e.g. all three from the extraction blank control from Ancient DNA Extraction from Skeletal Material - dx.doi.org/10.17504/protocols.io.baksicwe) and the library build control (e.g. from non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing dx.doi.org/10.17504/protocols.io.bakricv6) to assess the performance of the protocol and the level of background contamination. Consider these three extra samples in your calculations for buffer preparations. To take along the positive controls is optional after you know that the library preparation worked out. 

Additional Tips
It is recommended to prepare 10% more of the calculated volume of all mastermixes to compensate for possible pipetting error.

[GUIDELINES]
Working in an Ancient DNA Laboratory
- All steps of this protocol prior to PCR amplification should take place in a clean room facility specifically designed for ancient DNA.
- The researcher performing lab work should be dressed in correspondingly suitable lab-wear, such as:
   - full-body suit with hood (e.g., Tyvek)
   - hairnet
   - face mask
   - two pairs of clean gloves
   - clean shoes 
   - protective glasses
- Sample processing and buffer/master mix preparation should be carried out in separated work benches with integrated UV irradiation (e.g. Dead Air PCR work bench)
- Surfaces and equipment should be regularly decontaminated with e.g. bleach solution or Thermofisher's DNA AWAY (or similar) and irradiated with UV.

Please see the following for more detailed guidance: 

Llamas, B. et al., 2017. From the field to the laboratory: Controlling DNA contamination in human ancient DNA research in the high-throughput sequencing era. STAR: Science & Technology of Archaeological Research, 3(1), pp.1–14. Available at: https://doi.org/10.1080/20548923.2016.1258824.

Working in an Molecular Biology Laboratory
- From PCR amplification onwards, all steps takes place in a standard DNA-based molecular biology lab.
Please keep in mind the safety guidelines of your specific country and institution.
Recommendations include wearing of:
- lab coats
- closed shoes and trousers
- safety glasses
- nitrile or latex gloves

Definitions
Stock-aliquot refers to a personal 'stock' (e.g. in a 50ml Falcon Tube) of reagents you can use across multiple sessions of this protocol. An 'aliquot' refers to a sub-aliquot of the stock, that is used for a single session of this specific protocol.

Protocol Specific Guidelines
This protocol requires the use of two rooms - a dedicated PCR-free ultra-clean library building room and a standard molecular biology lab for qPCR.

[STEPS]
SECTION: Indexing preparation (aDNA library preparation room)
1. 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. 

SECTION: Indexing preparation (aDNA library preparation room)
2. Assign unique dual index combinations of index primer pairs to each library.
SECTION: Splits: 2 (aDNA library preparation room)
3. 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 78 µL mastermix, 2 µL of each index primer and 18 µLsample to each tube (use 0.2 mL PCR strips).

 

 

SECTION: Splits: 4 (aDNA library preparation room)
4. 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 87 µL mastermix, 2 µLof each index primer and 9 µL sample to each tube (use 0.2 mL PCR strips).

 

 




SECTION: Splits: 6 (aDNA library preparation room)
5. 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 90 µLmastermix, 2 µL of each index primer and 6 µL sample to each tube (use 0.2 ml PCR strips).

 

 

SECTION: Move to modern DNA lab
6. Securely close the reactions and transfer to modern DNA laboratory. If possible, keep the reactions on ice during the transfer. 
SECTION: Indexed adapter ligation with PCR (modern DNA lab)
7. In a modern DNA lab, use a thermocycler to amplify the reactions with the following program:

 

 

SECTION: MinElute Purification (modern DNA lab)
8. Purify the indexed libraries with a MinElute kit, with the following modifications to the manufacturer's protocol.
SECTION: MinElute Purification (modern DNA lab)
8.1. For each reaction, add 650 µL  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. 

 

SECTION: MinElute Purification (modern DNA lab)
8.2. Load each reaction (PB buffer + library) onto a MinElute column and incubate at RT for 2 min .

 

SECTION: MinElute Purification (modern DNA lab)
8.3. Spin at 15800 x g, 1 min  and discard flow-through.


 

SECTION: MinElute Purification (modern DNA lab)
8.4. Add 700 µL  PE (wash) buffer to the MinElute column.

SECTION: MinElute Purification (modern DNA lab)
8.5. Spin at 15800 x g, 1 min and discard flow-through.

SECTION: MinElute Purification (modern DNA lab)
8.6. Dry spin at 15800 x g, 1 min .

SECTION: MinElute Purification (modern DNA lab)
8.7. Remove columns from their collection tubes and place them in new 1.5 mL LoBind tubes.
SECTION: MinElute Purification (modern DNA lab)
8.8. Add 50 µL EBT to the column of the filter, let stand for 1 min, then spin at 15800 x g, 1 min to elute.

 

 

SECTION: qPCR Quality Check (modern DNA facility)
9. Dilute 2 µL 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. 

SECTION: qPCR Quality Check (modern DNA facility)
10. Prepare a qPCR assay calculating 20 µL. Prepare 2 reactions per sample, plus 16 additional reactions for 7 qPCR standards in duplicates and 2 qPCR blanks.


 

 

SECTION: qPCR Quality Check (modern DNA facility)
10.1. Add 19 µL mastermix and 1 µL diluted libraries, standard, or water for each reaction to a fresh 96-well plate.

 

SECTION: qPCR Quality Check (modern DNA facility)
11. Amplify the qPCR reactions with the following program: