Thanks to SPRQ chemistry and the Ampli-Fi protocol, researchers are now unlocking whole genomes from one of the most notoriously tricky sample types: dried blood spots (DBS). Collected from heel pricks in newborns, DBS have powered decades of screening programs worldwide. But for long-read sequencing, these samples have historically been a no-go because of their low input and low quality.
That’s changing fast.
A new breakthrough study showcases how HiFi sequencing can now deliver full, accurate genomes straight from DBS without short-read crutches or extra workarounds. We’re so excited to see how high-quality long reads are now creating new possibilities for rare disease discovery and clinical research in ways that weren’t possible before.
The value of newborn screening programs
Newborn screening (NBS) is a cornerstone of preventive medicine, designed to detect potentially serious but treatable health conditions in newborns, allowing timely intervention that can change the course of a child’s life. More than 60 genetic, metabolic, and endocrine disorders, including cystic fibrosis, can now be identified early through screening protocols. Central to this effort is the use of dried blood spots (DBS), which are collected via heel prick within days of birth.
DBS samples are ideal for logistics: they’re easy to collect, ship, and archive, and can be stored at room temperature for extended periods without significant degradation. Because of these characteristics, DBS has become the universal specimen type for newborn screening programs worldwide. Despite their widespread use, extracting high-quality genomic information from these samples has historically been a major technical hurdle, and genome sequencing is often only performed as a confirmatory test.
Challenges in sequencing DNA from dried blood spots
Though logistically ideal, this sample type can pose challenges for sequencing technologies that rely on longer, high-quality DNA fragments, especially long-read sequencing. As a result, most genome sequencing on DBS samples has been performed with short-read whole exome sequencing, which comes with its own set of limitations.
Short reads can miss structural variants, have trouble in repetitive parts of the genome, and are unable to phase variants – the ability to determine whether two variants are sitting on the same chromosome or not – a detail that can make all the difference in understanding a genetic condition.
HiFi sequencing vs. Illumina in overcoming dried blood spot barriers
Despite being the clear technology to overcome these challenges, long-read sequencing had been thought to be a nonstarter for DBS, given the nanogram-level DNA recovery typically achievable from heel prick sampling. But new innovations in HiFi sequencing are rewriting these assumptions. Transformative improvements in library prep and sequencing chemistry now make it possible to generate rich, clinically-relevant data from these minimal inputs. This shift opens the door for genome-wide phasing, structural variant detection, and accurate variant calling from dried blood spot-derived DNA.
At the forefront of this advancement is a recent study led by researchers at the Yale School of Medicine. This team performed whole genome sequencing (WGS) on two cystic fibrosis samples, one with a standard whole-blood sample and the other from a newborn dried blood spot. For this study, a single 3.2-mm dried blood punch was used for sample extraction. Using a precursor of the Ampli-Fi protocol, the researchers were able to sequence only 20 ng of input DNA with ~30x coverage on the Revio system in a head-to-head matchup with the Illumina Nova-Seq.
The results are profound. Like Illumina, HiFi sequencing identified pathogenic variants in the CFTR gene, but as a critical difference, only HiFi reads could successfully phase these variants, over 10.8 kb in the dried blood spot sample. Beyond exonic regions, HiFi sequencing also outperformed Illumina in accurately resolving complex or homopolymer-rich intron regions that are critical for comprehensive CFTR interpretation. Importantly, across both samples, HiFi sequencing identified pathogenic variants without the need for parental testing.
Transformative impact of HiFi sequencing with dried blood spots
As a critical benchmark, these researchers demonstrated consistent HiFi sequencing performance across both samples, confirming DBS as a robust sample type for HiFi WGS. Furthermore, with the ability to phase CFTR variants and resolve complex intronic regions, this study demonstrates the benefits of HiFi sequencing beyond sequencing of DBS using short-reads.
The success of this study sets a precedent for the broader use of HiFi WGS in challenging disease screening applications. It also opens the door to additional disease targets in newborn screening programs, greater yield, and more equitable access to more comprehensive sequencing information. Furthermore, eliminating the requirement for parental genotyping streamlines workflows and allows for much faster genetic resolution. This could make a profound difference in newborn screening applications where time is often critical.
Enabling the future of dried blood spot sequencing with Ampli-Fi and SPRQ chemistry
This breakthrough is just the beginning for HiFi sequency utility for DBS. The introduction of the Ampli-Fi protocol lowers the DNA input barrier even further, enabling library prep from as little as 1 ng of DNA. Coupled with improved SPRQ chemistry for the Revio system that not only reduces input requirements but also improves sequencing performance and yield, these advances represent the technological foundation for an inflection point in genome sequencing from dried blood spot samples.
HiFi sequencing is rewriting the narrative around what kinds of samples are considered viable for high-resolution genomic analysis. Where low-input samples like DBS were once thought to be unsuitable for comprehensive sequencing, innovations like Ampli-Fi and SPRQ are turning that assumption on its head. For newborn screening, this means the potential to gather accurate, genomic information earlier and with fewer limitations than ever before.
A new chapter for newborn screening genomics
The study highlighted here demonstrates that HiFi sequencing on the Revio system, combined with Ampli-Fi for ultra-low DNA input, can deliver the same full-genome phasing and variant calling from a single dried blood spot. In doing so, it unlocks the possibility of integrating HiFi sequencing into newborn screening workflows, eliminating the need for additional parental samples, reducing time to genetic resolution.
As innovations in HiFi sequencing continue to lower input requirements and drive down costs, HiFi WGS from DBS is poised to become a more comprehensive option for newborn screening. By making comprehensive sequencing accessible from routine sample types, HiFi technology is helping close the gap in early genetic screening, bringing us closer to a world where timely insight is possible for every newborn.
To learn more about the Ampli-Fi protocol and how it supports ultra-low input HiFi sequencing, check out our blog post here.