https://med.stanford.edu/gssc/hiseq4000issue.html
https://enseqlopedia.com/2016/12/index-mis-assignment-between-samples-on-hiseq-4000-and-x-ten/
If free barcoded adapter / index primers are present in a multiplexed pool, the free adapter has the potential to prime and extend library molecules in the same lane during the clustering step. This can result in mis-assignment of reads through index swapping. This can cause errors in demultiplexing data, as reads from one sample have the potential to end up in the FASTQ files of a different sample. The HiSeq 2000/2500 and MiSeq are less impacted due to their biochemistry and the geometry of the flow cell used.
The range of mis-assignment can vary significantly and is impacted by the following factors:
- Amount of free adapter present in library
- Storage conditions of library
- Application or library prep workflow
Sample mis-assignment can potentially impact users depending on the experimental design and library prep workflow. Illumina has been working on this issue internally and has developed a few suggested mitigation strategies to reduce index swaps, listed below:
During Library Construction:
- Optimize your PCR or ligation step to avoid an excess of adapters or index primers.
- For PCR dilute the index primers to adjust the insert to adapter / primer ratio.
- Perform extra clean ups after this step.
- PAGE purification seems to do a good job reducing indexing primers.
- Purification columns are also an option.
- Do extra clean ups of each individual library before pooling.
- Use single use aliquoted adapters and primers.
- Freeze individual libraries and pool prior to sequencing.
Pooling suggestions:
- Use dual indexing strategies with unique barcodes on both ends. (Swapping would have to occur at both ends for read mis-assignment to occur)
- Sequence or freeze created libraries pools as soon as possible.
Sequencing suggestions:
- Use PhiX from third parties with unique indexing barcodes to determine swap frequency. (We will have begun to introduce PhiX with unique barcodes from SeqMatic for HiSeq 4000 runs.)
- For methods highly sensitive to mis-assignment use HiSeq 2000/2500 or MiSeq instruments.
