For PacBio’s HiFi sequencing approach, pure high molecular weight DNA is key to successfully generating highly accurate long reads, also known as HiFi reads. DNA quality not only influences the length of the DNA molecules for the sequencing libraries, but also the overall output of PacBio’s HiFi sequencing.
DNA is susceptible to various forms of damage. For example, DNA samples can suffer damage and fragmentation due to
- multiple unnecessary freeze-thaw cycles,
- excessive pipetting,
- exposure to high temperatures, extreme pH values, intercalating fluorescent dyes, ultraviolet radiation, or
- breakdown of DNA due to the formalin fixation and storage in FFPE samples,
leading to low-quality DNA. Additionally, contamination with singlestranded DNA, RNA, proteins, dyes, or salts can influence the DNA quality and the overall sequencing result. During PacBio library preparation, hairpin adapters are ligated to both sides of the DNA fragments, creating a circular template for the polymerase.
After the library preparation and polymerase binding, the libraries are loaded onto a PacBio SMRT Cell for sequencing. Each SMRT Cell contains a fixed number of tiny wells known as zero-mode waveguides (ZMWs). In these ZMWs, single library molecules are immobilized, and nucleotide incorporation by the polymerase is measured and recorded in real time. The aim is to load the SMRT Cell in a way that one ZMW contains a single DNA library molecule. During the sequencing process, the circularized DNA is sequenced in repeated passes. After sequencing, the continuous polymerase reads are trimmed of the adapters, and a consensus sequence is generated based on all subreads. The consensus reads are filtered based on their quality, leaving only highly accurate, so-called HiFi reads. As a result, each occupied ZMW will produce one HiFi read, assuming it will pass quality filtering.
At a glance
- Low-quality DNA often has a higher degree of fragmentation, leading to shorter DNA fragments.
- Impurities and contaminations can inhibit or decrease the binding efficiency of the DNA-polymerase complexes.
- Average DNA size and SMRT Cell loading correlate with the overall SMRT Cell output.
- Good starting material and high-molecular weight DNA are essential for sufficient output and high-quality results.
As the number of ZMWs on a SMRT Cell is fixed, the generated output directly correlates with the size of the DNA molecules in the library and the loading of the SMRT Cell. Both parameters (average DNA size and SMRT Cell loading) are linked to DNA quality, as low-quality DNA often shows a high degree of fragmentation and can contain impurities that inhibit or decrease the binding of the DNA-polymerase complexes to the SMRT Cell. To illustrate this correlation, figure 1 shows a simplified version of the sequencing process, assuming we have a SMRT Cell with a total of 5 ZMWs and a sample with low-quality DNA (fragmented and containing impurities) versus a sample with pure high molecular weight DNA. The impurities in the low-quality sample might lead to reduced ZMW binding, resulting in loading only 2 of the 5 ZMWs, compared to the pure sample, where 4 ZMWs are loaded. Please note that SMRT Cells are generally never completely loaded in the PacBio workflow. Furthermore, we assume that the low-quality sample contains shorter DNA fragments (8 bp) than the high molecular weight DNA sample (16 bp). As each ZMW loaded with a DNA-polymerase complex will theoretically result in one HiFi read, the total output of the SMRT Cell is calculated by multiplying the number of loaded ZMWs (= HiFi reads) by the average DNA molecule size. As a result, the low-quality DNA will have a total base output of 8 bp x 2 loaded ZMWs = 16 bp versus 16 bp x 4 loaded ZMWs = 64 bp for the pure high molecular weight DNA.
Obviously, this is a fictitious example – but it highlights the importance and influence of the starting material on the output: Poor starting material with low-quality DNA will result in shorter HiFi reads and less output. Therefore, it is important to consider the starting material when estimating the output in gigabases. Using the number of HiFi reads might be a more robust estimation. Good starting material and high molecular weight DNA are essential for generating long libraries, good sequencing performance, and high-quality results. For this reason, we have prepared flyers with tips on DNA extraction and DNA preparation for PacBio’s HiFi sequencing.
Figure 1 | Influence of the starting material on the output. Samples with low-quality DNA can yield shorter DNA fragments, resulting in short sequencing libraries. Additionally, the impurities in the low-quality sample might lead to reduced ZMW binding. Consequently, fewer and shorter HiFi reads are generated during the sequencing process compared to samples with high molecular weight DNA, leading to a reduced output.