How do I amplify Illumina sequencing libraries?

In case the library preparation did not generate sufficient library material required to load a sequencer, Illumina libraries can be amplified with a universal PCR protocol.
While the amplification can rescue experiments, it is worth considering on a per-project basis if perhaps the library preparation should be repeated instead. For quantitative experiments, it is generally recommended to treat all libraries the same throughout the pipeline. Insufficient library yields in the initial library preparation could be signs of sample contamination, processing errors, etc. with potential side effects that cannot be remedied by amplification.

Illumina library amplification PCR protocol:
The library amplification uses standard Illumina P5 (5′-AATGATACGGCGACCACCGAGATCT-3′) and P7 (5′-CAAGCAGAAGACGGCATACGAGAT-3′) PCR primers which can be ordered as desalted DNA oligos.
Create a 10x concentrated primer mix at 10 μM each of these primers in EB buffer.

Add up to 20μl library, add water up to a volume of 20μl, add 5 μl 10x primer mix. Then add 25μl Kapa HiFi 2x Hotstart PCR master mix and pipette up and down several times.
Use the following cycling parameters:
Initial denaturation: 98°C 45 sec,
X PCR amplification cycles consisting of: denaturation 98°C 15 sec, annealing 60°C 30 sec, extension 72°C 30 sec
Final extension 1 min.

Assuming one wants to generate at least 100 ng of sequencing library, we recommend performing four cycles of PCR when starting from 10 ng library.
After the PCR perform a standard Ampure XP/SPRI bead cleanup (e.g. with beads at 1.2x the sample volume, Ampure beads or equivalent) and elute in 30μl EB buffer.

Category: 04 Library Preparation and QC

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