February 1, 2016 (Vol. 36, No. 3)

Radmila Hrdlickova Senior Scientist Bioo Scientific
April Lewis Bioinformatician Bioo Scientific
Jiri Nehyba Senior Scientist Bioo Scientific

New Technology Enables High Uniformity and Multiplexing Capabilities

Targeted sequencing is an economical way to obtain sequencing reads that are limited only to genomic regions of interest. There are two different approaches used in targeted sequencing—exome capture and amplicon sequencing.1,2 While exome capture has advantages for whole-exome sequencing (30 Mb for the human genome), amplicon libraries offer more specific, cost-effective results in a shorter time and are ideal for use when the targeted regions are smaller than 200 kb.3

In many research and diagnostic scenarios, the sequencing of a limited number of genes is required for a large number of samples. Amplicon libraries are the method of choice in these scenarios.

There are two major categories of DNA amplicon libraries—those used for the screening of germline mutations and those used for the screening of somatic mutations. The screening of germline mutations does not require deep sequencing, and high-quality input material typically is available. These panels usually contain a low number of amplicons that, when amplified with high uniformity, allow for a high level of multiplexing and economical sequencing.

Non-uniform coverage in amplicon panels necessitates more reads per panel, which can dramatically increase sequencing costs. Amplicon panels for the screening of somatic mutations require deeper sequencing and often must perform well with partially degraded samples. From an economic standpoint, high uniformity is imperative because the detection of rare mutations (with an abundance below 5%) depends on high coverage.

Recently, demand has also developed for panels that enable the screening of mutations introduced by gene editing. These panels consist of just a few amplicons; however, a great number of samples are sequenced simultaneously. Distinguishing these samples requires the introduction of thousands of specific labels.

There are two methodological problems with the development of amplicon panels:

  1. Amplicon panels need to be designed with high uniformity in order to permit even coverage when a high depth of sequencing is the goal. Uniformity is most critical for large panels that screen somatic mutations in degraded DNA samples. The uniformity of the amplicon panels is a function of the design of the target-specific primers.
  2. Certain applications of amplicon sequencing also require a high level of multiplexing. Amplicon panels with fewer than 200 amplicons, which are used for germline disease predisposition testing based on family history, as well as the screening of introduced mutations, benefit from high multiplexing capabilities.

Figure 1. Schematic workflow of primer design for amplicon panels using Bioo Scientific primer design software.


Bioo Scientific recently introduced NEXTflex™ Custom Amplicon Panels, which offer both high uniformity and high multiplexing capabilities. Primer design is based on a scoring system that uses machine learning to evaluate each possible primer according to an individual score (Figure 1). The score is calculated as a sum of the weighted values of multiple parameters.

The results of the performance of the panel, obtained by library preparation and sequencing, are then correlated with the predicted value of the amplicon primer pair. The correlation analysis results are used to modify the primer design software by manipulating the parameter weights or by creating new parameters that better correlate with amplicon performance.

The new software setting will be used in subsequent primer design to optimize primers for an individual researcher’s needs. The ability to modify the software’s settings allows optimized design strategies to be used for regions containing challenging sequence compositions or for different library preparation methods.

A quality check (QC) option, which evaluates primers designed manually or by different primer design engines, is also included. This primer design software is used to develop Bioo Scientific’s custom and fixed panels. The performance results obtained during amplicon panel testing are used continuously to design higher-precision primers with uniform performance.

Typically, for the Illumina sequencing platform, 96 barcodes are available for multiplexing. However, higher levels of multiplexing are necessary for some amplicon panels. To meet this demand, Bioo Scientific offers a 384-amplicon barcode system. Moreover, for projects in which thousands of samples must be analyzed by a few amplicons and sequenced simultaneously, NEXTflex has developed Custom Amplicon Panels that are tagged with a combination of specific amplicon primers and the set of 384 barcodes.

Figure 2. Application of primer design software. (A) Correlation between amplicon scores (Fscore) obtained with unweighted and weighted parameters and amplicon performance. (B) Performance of 131 amplicons from BRCA1 and BRCA2 panels for detection of germline mutations.


The primers used for library preparation during the development of the BRCA1 and BRCA2 panels were scored by QC, which is part of Bioo Scientific’s proprietary primer design software. The correlation between primer performance and score demonstrates the prediction value of primer scoring (Figure 2A). When all weights are set at 1, correlation is low. When the weights are adjusted based on amplicon performance, the scoring system can design primers for highly uniform panels. Without weighting, the worst-performing amplicons cannot be predicted, but the introduction of weight coefficients facilitates the identification of low-quality sequence primers. This method is flexible and allows for the optimization of primer design for different library preparations, target regions, or panel characteristics.

The NEXTflex BRCA1 and BRCA2 Amplicon Panels for germline mutations were developed using the approach described in this article. The uniformity of these panels is 100%, which means that all amplicons are performing better than 20% of the average. Moreover, the best panel exceeds the industry standard, with all amplicons performing better than 40% (Figure 2B). This high uniformity would allow the sequencing of 700–900 libraries per MiSeq run, with 100× coverage of every region.

Figure 3. Schematic representation of the principle behind the high-multiplexing system developed by Bioo Scientific.

For deep multiplexing, Bioo Scientific uses a set of 384 3′ adapters with 12 nt indexes, which are introduced during the PCR amplification step of amplicon library construction. These 12 nt indexes have a Hamming distance of at least five, permitting dual error correction. For the multiplexing of more than 384 samples with panels of a limited number of amplicons (e.g., the sequencing of a single gene), additional short 3 nt tags with a Hamming distance of three are introduced with the amplicon-specific primers (Figure 3). These tags do not introduce any bias to the amplification of specific gene regions (Table). The combination of three 5′ and three 3′ tagged primers increases the multiplexing power nine-fold, enabling the sequencing of 3,456 libraries in a single well.


To improve amplicon targeting, the NEXTflex Custom Amplicon Panels incorporate two novel technological elements: optimized primer design software and the ability to multiplex up to 3,456 libraries for sequencing. The unrivaled uniformity and flexibility of primer design offered by the NEXTflex Amplicon Studio makes amplicons ideal for the detection of both germline and somatic mutations. This highly targeted approach offers unparalleled efficiency for discovering, validating, and screening genetic variants.

1. Clark MB, Mercer TR, Bussotti G, Leonardi T, Haynes KR, Crawford J, Brunck ME, Cao KA, Thomas GP, Chen WY, Taft RJ, Nielsen LK, Enright AJ, Mattick JS, Dinger ME. 2015. Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing. Nat Methods 12:339-342.
2. Zhang JD, Schindler T, Kung E, Ebeling M, Certa U. 2014. Highly sensitive amplicon-based transcript quantification by semiconductor sequencing. BMC Genomics 15:565.
3. Samorodnitsky E, Jewell BM, Hagopian R, Miya J, Wing MR, Lyon E, Damodaran S, Bhatt D, Reeser JW, Datta J, Roychowdhury S. 2015. Evaluation of Hybridization Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing. Hum Mutat 36:903-914.

Radmila Hrdlickova ([email protected]) is senior scientist, April Lewis is bioinformatician, and Jiri Nehyba is senior scientist at Bioo Scientific. To learn more about the NEXTflex Custom Amplicon Panels, visit www.Biooscientific.com/panels.

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