In other words, depending on the sequence size and the power of the machine available, it is possible to perform the analysis of more than one sample at a time. It is not uncommon, for example, to perform the exome sequencing for two individuals in a single run. In microbiology for instance because the genomes of microorganisms are very small, it is possible to sequence the genomes of many viruses and bacteria simultaneously.
The analysis of multiple samples in parallel is called multiplexing. In order to do a multiplexing analysis, it is necessary to add to each library fragment a short, patient-specific, synthetic sequence, also known as barcode sequence. The barcode sequence works as a label to uniquely identify all the DNA fragments belonging to the same individual (or to the same microorganism). Once the DNA library fragments of every sample have been labelled with the barcode sequence, they can be put all together into the same tube to start the sequencing reaction. All the reads produced will also contain the barcode sequence. Thanks to the barcode sequences it will be then possible to separate the reads belonging to each sample (de-multiplexing) before proceeding to alignment and variant calling.
Multiplexing became a reality just thanks to NGS, since costs and times have always made such analysis prohibitive by means of Sanger capillary electrophoresis. NGS machines also minimize the human intervention in the analysis, since there's no electropherogram to interpret and alignment and variant calling are fully automated. Not only that: the amount of DNA required for an NGS analysis (even in multiplexing) is much lower than what is needed for a Sanger analysis: only 30 ng of DNA may be enough to get an entire exome!
Related articles:
- EXOME SEQUENCING
- NEXT GENERATION SEQUENCING FOR DUMMIES
- NEXT GENERATION SEQUENCING: APPLICATIONS
- REFERENCE SEQUENCE: WHAT IS IT?
