This information is a stub to be added into a reworked NGS protocol as an alternative. Follows the duplex seq method developed by the Loeb lab

This protocol is for rare variant applications. It is a waste of time, money, and reagents to use this protocol on samples which you do not expect and/or are not interested in mutations that exist at a frequency of <1%. Consider which protocol is best before proceeding. Please click here for standard protocol.

Adapter Annealation

Note, this step is only necessary if 'Annealed Adapters' tube is empty. This should be a rare step. This step can be scaled up, but some evidence that after ~14-18 months annealed extended adapters don't function as well, so don't make too much excess.

1. Mix the following in a PCR tube:
   • 20µl of Adapter_forward (100pmol/µl)
   • 20µl of MI_Reverse (100pmol/µl)
2. Use thermocycler with following conditions to anneal the 2 primers together:
   1. 97C 2minutes
   2. 97C 1minute
      1. Repeat previous step for 72 total cycles at -1C per cycle
   3. 25C 5 minutes
      After this step, adapters should NEVER be warmed above 25C.
3. To the annealed adapters, add the following extension reaction reagents:

Item	Amount
WFI water	5.5µl
10x NEB 2 Buffer	6µl
2.5mM dNTP	6µl
Klenow exo- (5 units/µl)	2.5µl

1. Incubate 1 hr at 37°C in thermocycler.
2. Ethanol precipitate by adding the following. Note the times and spins of this precipitation are excessive, but they are an attempt to increase recovery as the adapters are quite expensive:

Item	Amount
Glycogen (20µg/µl)	2µl
NaOAC (3M pH 5.2)	6.2µl
100% EtOH	170.5µl

1. Mix well and precipitate overnight at -20°C
2. Spin at max speed for 30 minutes at 4°C.
3. Decant carefully and/or pipette without disturbing pellet.
4. Add 1mL 70% EtOH and resuspend via flicking tube.
5. Spin at max speed for 30 minutes at 4°C.
6. Decant carefully and/or pipette without disturbing pellet.
7. Air dry 10 minutes inverted sitting on fresh kimwipe.
8. Air dry 5 minutes upright near burning flame.
9. Resuspend in 40µl of WFI water.
10. dA tail the adapters by adding the following:

Item	Amount
WFI water	5.5µl
10x NEB 2 Buffer	6µl
10mM dATP	6µl
Klenow exo- (5 units/µl)	2.5µl

1. Incubate 1 hr at 37°C in thermocycler.
2. Ethanol precipitate by adding the following. Note the times and spins of this precipitation are excessive, but they are an attempt to increase recovery as the adapters are quite expensive:

Item	Amount
Glycogen (20µg/µl)	2µl
NaOAC (3M pH 5.2)	6.2µl
100% EtOH	170.5µl

1. Mix well and precipitate overnight at -20°C
2. Spin at max speed for 30 minutes at 4°C.
3. Decant carefully and/or pipette without disturbing pellet.
4. Add 1mL 70% EtOH and resuspend via flicking tube.
5. Spin at max speed for 30 minutes at 4°C.
6. Decant carefully and/or pipette without disturbing pellet.
7. Air dry 10 minutes inverted sitting on fresh kimwipe.
8. Air dry 5 minutes upright near burning flame.
9. Resuspend in 40µl of WFI water.
10. Make sure tube is well labeled.
11. Store -20°C

T-Tailing Reaction

1. This step uses NOTHING from the kapa kit besides the PEG/NaCL solution.
2. Make sure heat block is set to 30C and holding.
3. Pull PEG/NaCL SPRI Solution and place on bench to warm to room temperature
4. To each tube add the following:

Item	Amount
WFI water	19.5µl
10x NEB buffer 2	2.5µl
10mM dTTP	2.5µl
Klenow exo- (5units/µl)	0.5µl

1. Mix by pipetting till homogeneous solution
2. Incubate 30 minutes at 30C.
3. Remove tubes from block.
4. Remove block from base, and place on bench in cold room. Set block to 20C. Check back periodically (at least 15 minutes needed) for block to stay at or below 20C once back in base. Ice buckets can be used instead of cold room bench for better heat dispersal.

PCR Addition of EBC

add note about number of cycles

-- Main.DanielDeatherage - 29 Apr 2015