Multiplexing (pooling) is a technique used to simultaneously analyze multiple samples in a single Next Generation sequencing run. It involves the use of unique index (barcode) sequences that are added to each sample before sequencing. These indexes/barcodes allow the samples to be distinguished from one another during the sequencing process.
One common application of multiplexing is for targeted sequencing, where only specific regions of the genome are sequenced. This is achieved by adding unique indexes/barcodes to each sample and then pooling the samples together for a single run. This can be useful for studying specific genes or genetic Regions of Interest (ROI) that are associated with a particular disease or trait.
Another application multiplexing is for whole genome sequencing, where the entire genome is sequenced. Again, this is accomplished by adding unique indexes/barcodes to each sample and then pooling samples. This allows for the simultaneous sequencing of multiple samples, which can be useful for studying population genetics or for identifying genetic variations in a specific disease or trait.
There are numerous benefits in multiplexing samples including:
- Increased sample throughput: Multiplexing allows for the simultaneous analysis of multiple samples in a single sequencing run, which increases the overall sample throughput and can save time and resources.
- Reduced cost per sample: Since multiple samples are being analyzed in a single run, the cost per sample is reduced compared to analyzing each sample individually.
- Improved experimental design: Multiplexing allows for the simultaneous analysis of multiple samples or conditions in a single experiment, which can improve the statistical power of the study and provide a more comprehensive understanding of the system being studied.
- Greater flexibility: Multiplexing allows for the simultaneous analysis of different types of samples or regions of interest, which can be useful for studying different aspects of a biological system or for identifying genetic variations associated with a specific disease or trait.
- Better data quality: Multiplexing allows for the simultaneous analysis of multiple samples, which can improve the overall data quality by increasing the number of replicate measurements and reducing the effects of technical variability.
- Greater scalability: Multiplexing allows for the simultaneous analysis of large numbers of samples, which can be useful for studying populations or large-scale genomic projects.
In most sequencing runs we multiplex or pool several or many samples to significantly reduce the cost per sample, to improve throughput in the Lab and to improve turnaround time on projects.