TITLE:         Amplification and Pooling 
AUTHORS: Franziska Aron, Guido Brandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the amplification procedure of dual-indexed double-stranded DNA libraries, for shotgun Illumina sequencing. It is typically used for libraries indexed using the following protocol: (</span><a href="https://dx.doi.org/10.17504/protocols.io.bakticwn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://dx.doi.org/10.17504/protocols.io.bakticwn</span></a><span style = ":;">)</span></div></div>

[STEPS]
?. [Calculations]
Prepare amplification assay [ ]Based on the quantification results of the indexed libaries (https://dx.doi.org/10.17504/protocols.io.bakticwn) calculate the number of PCR cycles (amplification factor) needed to reach 10^13 copies of DNA per indexed Library.Example: The following calculation is for  per reaction, with two indexed library samples (A and B) having different concentrations of DNA copies.Optonial Changes: 1.If the Calculation shows up less then 3 Cycles, you also have the Option to add less then 5 µl.                                   2. Instead of 4 reactions of 5 µl each you can also split in 8 reactions of 2 µl each Instead of 4 x 5 µl reactions you can also split in 8 x 2 µl reactions  ABCDEFGH1Sample NameCopies per µlµl per rxnCopies per rxnCycles neededReal CyclesAmplification FactorOutput per rxn [Copies]2A7.32E+1053.66E+114.7729985321.17E+133B5.79E+0652.32E+0718.72018251195242881.21E+13
[per reaction]
Formula in Excel to get the Cycles needed=LOG((1*10^13/Copies per rxn),2)(log base 2)
5 µl
ABCDEFGH1Sample NameCopies per µlµl per rxnCopies per rxnCycles neededReal CyclesAmplification FactorOutput per rxn [Copies]2A7.32E+1053.66E+114.7729985321.17E+133B5.79E+0652.32E+0718.72018251195242881.21E+13
Do not calculate the amount of cycles for a higher amount of copies than 1.4 *10^13 to avoid heteroduplexes.
?. [PCR]
Amplify in a thermocycler with the following program:  ABC1TempreatureTime295°C2 minInital denaturation395°C30 secCycles (see Step 1)460°C30 sec572°C30 sec672°C5 minFinal elongation7 Finally hold the reactions at 10 °C.
ABC1TempreatureTime295°C2 minInital denaturation395°C30 secCycles (see Step 1)460°C30 sec572°C30 sec672°C5 minFinal elongation7 Finally hold the reactions at 10 °C.
Adjust the number of cycles according to the amplification factor as calculated in step 1.During this incubation take MinElute columns out of the fridge so they warm up to room temperature before use in the next step. This is an ideal point to prepare downstream steps, including labelling of final elution tubes, MinElute Columns etc.
?. [MinElute Purification]
Purify with MinElute kit with the following modifications to the manufacturer's protocol:Use one column for all four reactions [= PCR product] of a sample.
400 µl
?. [Measurement and Dilution]
Dilute amplified index library 1:10 with HPLC- water and check for fragment size, concentration, and heteroduplexes. (for example with the D1000 Kit's Tape, Reagent and Buffer - following the manufacturer's protocol on the TapeStation)
if you see heteroduplexes you need to perform a reconditoning PCR. Reconditoning PCR: one cycle PCR using 100 ng library template in a 100 µl Herculase PCR reaction (same set up as in 3) and amplified with 1 cycles of 95°C for 2 min, 58°C for 2 min, and 72°C for 5 min. Purify with MinElute kit following the instructions from Step 5, but elute in 20µl EBT.
?. [Measurement and Dilution]
Dilute each amplified indexed library to  with EBT buffer or HPLC-water for shotgun sequencing. Then pool the  amplified indexed libraries in equimolar amounts (take the same volume for each sample).
10
10
The final concentration of a pool of several amplfied indexed libraries should be  .
?. [PCR]
Set up four amplification reactions of  each per library   ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2Herculase II Reaction buffer5x1x203IS5 primer10 µM0.4 µM44IS6 primer10 µM0.4 µM45dNTP's25 mM0.25 mM16Herculase II Fusion1 U0.01 U17DNA58HPLC-Water659Total100
100 µl
ABCD1ReagentStock concentrationFinal concentration1x Volume [µl]2Herculase II Reaction buffer5x1x203IS5 primer10 µM0.4 µM44IS6 primer10 µM0.4 µM45dNTP's25 mM0.25 mM16Herculase II Fusion1 U0.01 U17DNA58HPLC-Water659Total100
?. [PCR]
Vortex master mix before adding the enzyme. After adding the enzyme, mix by pipetting or inverting the tube.
?. [PCR]
Pipette  mastermix and  indexed library into each tube (use  PCR strips).
95 µl
5 µl
0.2 mL
Keep the remaining library at  until further use.
?. [MinElute Purification]
Add  PB or PBI* buffer to a   tube for each sample (this is buffer for each PCR reaction). Add all 4 PCR reactions per sample to the same tube with PB buffer and vortex briefly.
2400 µl
5 mL
600 µl
After the PCR product is mixed with the PBI, the PBI should keep its yellow colour. If it turns purple the pH is too high and the efficency of the MinElute columns is not guaranteed.
?. [MinElute Purification]
Add  PE (wash) buffer,  spin , and discard flow-through.
700 µl
Centrifuge: 15800 34, 1 min
?. [MinElute Purification]
Add  EBT buffer to the center of the filter, incubate for , and spin  to elute the amplified indexed library.
50 µl
Centrifuge: 15800 34, 1 min
Carefully pipette EBT directly onto the center of the membrane without touching the membrane.
?. [MinElute Purification]
Repeat loading until the complete mixture was run through the column.
?. [MinElute Purification]
Load  of the mixture onto one MinElute column, incubate for , spin , and discard flow-through.
700 µl
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]
Put column into new   LoBind tubes.
1.5 mL
?. [MinElute Purification]
Dry spin ,
Centrifuge: 15800 34, 1 min
?. [Measurement and Dilution]
Check the  library or the  library pool for the correct concentration, (for example with the HighSensitivity D1000 Kit's Tape, Reagent and Buffer following the manufacturer's protocol on the TapeStation.)
10
10
?. [Preparation]
Prepare cleaned workspace with all necessary reagents and equipment.
Label all    PCR strips for the PCR reactions.