To reduce costs of NGS, libraries are often prepared using molecular barcodes, also known as indices. The NEXTflex™ Barcodes contain index sequences, short sequences of 6 – 12 bases that are incorporated into each library to allow several libraries to be multiplexed and sequenced together on a single flow-cell of a sequencer. When barcoded NGS libraries are sequenced, each cluster generated on an Illumina sequencer includes its own unique barcode sequence, which is then associated with a specific library. Barcodes can be introduced into the library during the adapter ligation step (by including them as a subset of the adapter sequences), or they can be introduced during the subsequent PCR step, by including them as a subset of the Forward or Reverse PCR primers. For DNA and mRNA library prep Bioo Scientific recommends introducing the barcodes during the adapter ligation step of library construction. This avoids problems due to amplification bias caused by PCR primer sequences, and allows use of multiplexing when making libraries that do not include a PCR step.

Low-plex multiplexing (i.e. mixing up to ~ 12 samples) is often chosen as a reasonable compromise between low cost/low per sample coverage, and high cost/high coverage. High levels of multiplexing can involve use of 96 or more different barcodes, usually using commercially available barcode sets such as Bioo Scientific’s NEXTflex-96™ Barcodes. With high level multiplexing, sufficient diversity in the large library of barcode sequences is ensured. In contrast, when using low-level multiplexing, it is possible that the barcode sequences chosen could lack sufficient diversity to avoid “registration failure” on an Illumina sequencer. Registration failure could occur if the color balance was not maintained between the red and green lasers (used to sequence A/C bases and G/T bases, respectively). Barcode sequences for low-level multiplexing need to be chosen such that each position of the barcode will result in signal in both the red and green color channels. In other words, each position in the set of index sequences needs to include at least one A or C base, at least one G or T base, and ideally an equal balance of both.


Examples of acceptable combinations of barcodes for 2-way multiplexing:


(every position in each pair has at least one A/C and one G/T)

Examples of unacceptable barcode combinations for 2-way multiplexing:


(first pair lacks G/T at positions 2 and 3 and lacks A/C at position 6; second pair lacks G/T at first position, lacks C/A at fourth and fifth positions)


With these considerations in mind, Bioo Scientific recommends the use of specific barcode combinations for low-level multiplexing. These low-level multiplexing recommendations can be found in Appendix A in all of our barcode manuals. Note that recommendations are given for low-plex barcode combinations even for our larger barcode sets, to provide flexibility for use in situations where low-plex multiplexing is desirable. – Dr. Marianna Goldrick