During next-generation sequencing library preparation, adapters are attached to each sample. These adapters are essential as they enable the sequencing library to adhere to the flow cell’s surface. However, not all adapters are the same. Different designs and combinations are available. This text outlines relevant considerations about these short sequences.
We will start with a few general thoughts about adapters. Adapters are short DNA molecules with a length of around 80 bases. They are flanking both sides of the sample fragment, the so-called insert, of a sequencing library molecule. The basic structure contains three main components. Firstly, the already mentioned flow cell binding sequence. This sequence is platform-specific. Secondly, the primer binding site. The sequencing primers bind to these regions, which ensures oligosynthesis by the polymerase. Both components, the flow cell binding side, and the primer binding side, are not variable. However, the third component needs to be unique – the individual index enables the unique tagging of a sample. This step is called barcoding, allowing several samples to be pooled and sequenced in a single run. In the subsequent bioinformatic analysis, barcoding prevents the samples from being mixed up.
Now that the three main components of an adapter have been described, let’s have a look at figure 1. It shows examples of different adapter designs. With a finished sequencing library, the adapter’s primer binding sites (SP1 and SP2) bind directly to the insert. The individual index regions i5 and i7 follow. The flow cell binding sequences (P5 and P7) are located outside of the adapters. The main distinction in the adapter design is the number of index sequences added. One distinguishes between single index and dual index adapters.
Figure 1 | Examples of library construction with different adapter designs. Libraries with a single index adapter design have an index sequence on only one side of the insert, while a dual index adapter design requires index sequences on both sides of the insert.
Single index adapters only contain one index sequence (i7). The i7 index is sequenced after the forward read during the sequencing process. Up to 48 unique sequences are available, making it possible to generate up to 48 libraries. In contrast, dual index sequencing includes two index sequences, as the name suggests. In addition to an i7 sequence, an i5 sequence is added. The recommended workflow is a unique dual index workflow. This means libraries have distinct, unrelated index sequences for both index reads. This method allows for the unique barcoding of up to 96 samples. A second dual-index sequencing option is called combinatorial dual-index sequencing. In this case, libraries share either the i7 or the i5 sequence.
What needs to be considered when choosing the right adapter design for your sequencing project? As only one index sequence is used with single index adapters, this method is limited to the number of unique tagging options. Therefore, this method suits smaller projects with fewer samples that need to be sequenced together. Yet, in addition to the increasing number of possible individually markable samples, the complexity of the dual index barcoding system also results in a higher accuracy of read identification after sequencing. This advantage is especially striking when using unique dual index barcoding as both indices, i7 and i5, differ for each sample. For many samples, this dual index barcoding approach minimizes the possibilities of misassignments between sample reads.
To summarize, adapters are the molecules ensuring your sample can be sequenced on a respective flow cell. Further, the adapters allow the individual barcoding of a sample. The barcoding can be performed with single or dual index adapters, resulting from different designs of adapter sequences. Which adapter best suits your project depends on the number of samples you want to analyze. Find the right one and start your sequencing project!