The ability to multiplex many samples on the same run makes Illumina sequencing a powerful and affordable tool for many researchers. When pooling samples for sequencing it is important that they are pooled at equal molarity to prevent over or under representation of any individual sample in the same run. Care also has to be taken in deciding how many samples can be combined to achieve the required sequencing depth, a factor that is further complicated in viral sequencing where the majority of the reads may originate from the host and only a small proportion corresponding to viral reads.
Also of major importance is the final pool quality control (QC) to ensure that all contaminating adapter dimers are removed, generate accurate size distribution and quantification of the final pool. Accurate QC ensures accurate loading on the sequencer, overestimation of the pool molarity can cause under clustering resulting in fewer reads and can even cause run failure. Underestimation of pool molarity causes over clustering, lowering the read quality and risking run failure.
Adapter dimers are small fragments containing full length adapter sequences which can bind to the flow cell and cluster. Due to their small size, they cluster more efficiently than the longer library fragments and so reduce the library-specific read depth as well as causing over clustering which reduces the data quality possible run failure. In addition free adapter can be incorporated into the library clusters resulting in index hopping and incorrect assignment of library barcodes.
Here we describe our standard workflow for determining the appropriate sequencing depth and pooling by equal molarity of Illumina sequencing libraries, along with our procedure for pool QC.