May 23, 2022  |  Human genetics research

Get more for less —
Single-gene testing with HiFi sequencing

Is it possible to save time and resources while also increasing the quality of data you receive? For many, this might seem impossible. But recently, some researchers have found that with targeted, single-gene sequencing using HiFi long reads, this is a possibility.

The search for efficiency and the benefits of HiFi sequencing

In today’s landscape, many private and public labs are driving towards workflow efficiency. Researchers are looking for ways to streamline operations by taking existing lab processes, consolidating them, and using newer technology to reduce costs and increase efficiency to achieve the results they need. Faster turnaround times, reduced staffing, and reduced equipment investment can all result in significant savings. This has led many researchers to use HiFi long reads for targeted, single-gene sequencing for consolidated testing modalities, higher throughputs, lower costs, and more critical information.

Historically, laboratories have had to use a combination of technologies including Sanger sequencing, MLPA, short read sequencing, and qPCR. Each of these technologies delivers different information required for a complete analysis of some of the most challenging, but medically relevant, genes in the human genomes. Luckily, PacBio’s highly accurate, true long-read HiFi sequencing allows laboratories to consolidate their disparate technologies onto one platform to provide complete insights for these genes. With HiFi sequencing, researchers can access the entire gene, including complex structural variants, phasing, and genetic context around variants of interest as well as discovering new ones.

Single-gene sequencing with true long reads — the results seen around the world

The benefits of optimized workflows can be seen firsthand in the work of Dr. Vorasuk Shotelersuk of Chulalongkorn University in Thailand. As featured in the April 2022 edition of the Journal of Clinical Endocrinology and Metabolism paper titled, “Long-read amplicon sequencing of the CYP21A2 in 48 Thai patients with steroid 21-hydroxylase deficiency,” Dr. Shotelersuk endeavored to look at cases of congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency (21-OHD) – an autosomal recessive disorder resulting from biallelic pathogenic variants (PVs) in the CYP21A2 gene. He wanted to see if long-read technology could be leveraged to help increase the solve rate in subjects with 21-OHD and potentially implement a new paradigm for newborn screening throughout Thailand.

To do this, Dr. Shotelersuk explored the possibility of simplifying his team’s workflow to get more data using a long-read sequencing approach. He implemented a new workflow with two key goals in mind:

  • Detect pathogenic variants in the CYP21A2 gene
  • Determine the workflow’s effectiveness in subjects already diagnosed with 21-OHD

The team’s approach resulted in a 100% concordance rate in cases of confirmed affected subjects, highlighting the opportunity to consolidate conventional molecular techniques without compromising performance. This is a great example of how long-read sequencing can potentially be used to further simplify newborn and carrier screenings.

Dr. Shotelersuk is not the only researcher finding success. In China, a team at Berry Genomics Corp. also looked at a newborn screening test. In a recently published paper, “Long-Molecule Sequencing: A New Approach for Identification of Clinically Significant DNA Variants in α-Thalassemia and β-Thalassemia Carriers,” the team shared how they approached the multiple molecular tests currently used to test for thalassemia, an inherited blood disorder, to develop a scalable, accurate and cost-effective genotyping method that could one day be used for large-scale carrier screening of the disorder.

Additionally, led by Dr. Sarah Charnaud published “PacBio long-read amplicon sequencing enables scalable high-resolution population allele typing of the complex CYP2D6 locus“, a study of a gene involved in the metabolism of 25% of commonly used pharmaceuticals. It was the largest PacBio sequencing cohort to date, leveraging HiFi sequencing for CYP2D6 to develop an analysis pipeline to characterize hundreds of Solomon Islanders, a previously uncharacterized population. The researchers identified several functional rare and novel alleles that change metabolizer status unique to the Solomon Island population. Their research highlights the feasibility of CYP2D6 for population scale analysis and the importance of complete genomic information to capture relevant variants in diverse populations.


Full length amplicon sequencing of CYP2D6
An Integrative Genomics Viewer image highlighting full-length reads across the CYP2D6 region. Over 40 heterozygous variants in an 8.1 kb CYP2D6 amplicon region are fully phased and allow for unambiguous haplotype resolution. Top reads (blue) correspond to the *9 haplotype and bottom reads (green) to *35.

Product updates to streamline single-gene sequencings


DNA to TargetWhile these examples highlight ease of use and efficiency, they are a small subset of the ways researchers are using targeted, single-gene sequencing to save time and resources by consolidating assays on the PacBio platform. Recently, we announced the launch of the SMRTbell prep kit 3.0 and flexible barcoding. Through this launch, researchers now have access to universal protocols and reformatted library and sequencing reagent kits that reduce workflow steps and complexity, saving at least 1 hour for SMRTbell library prep of amplicons for sequencing on the Sequel IIe system.

With PacBio long reads you can easily and cost-effectively sequence full length amplicons that target genes or regions of interest, from several hundred base pairs to kilobase scale. Highly accurate long reads allow you to target all types of variation, from single nucleotide variants and indels, to structural variants. HiFi reads can reveal biology that other technologies miss, like low complexity regions and repeat expansions, GC-rich promoter regions, and insertion sites of transposable elements.



Barcoded primers and adapters

Interested in learning more about what you can do with long-read sequencing?

Visit the Targeted sequencing applications page to learn more.

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.