Improvements in next-generation sequencing (NGS) technology have greatly increased sequencing speed and data output, resulting in the massive sample throughput of current sequencing platforms. Ten years ago, the Genome Analyzer was capable of generating up to 1 Gb of sequence data per run. Today, the NovaSeqTM 6000 System, built on the same core technology, is capable of generating up to 2 Tb of data in two days, which represents a > 2000 increase in capacity.
A key to utilizing this increased capacity is multiplexing, which adds unique sequences, called indexes, to each DNA fragment during library preparation. This allows large numbers of libraries to be pooled and sequenced simultaneously during a single sequencing run. Gains in throughput from multiplexing come with an added layer of complexity, as sequencing reads from pooled libraries need to be identified and sorted computationally in a process called demultiplexing before final data analysis (Figure 1).
