Technical note — High throughput DNA shearing for HiFi whole genome sequencing from whole blood samples
AMPure® PB bead size selection is a simple, quick, and scalable approach to deplete molecules less than 5 kb in a SMRTbell® library. For high-quality DNA, the sequencing performance and application metrics for assembly and variant calling are comparable to gel cassette size-selection methods.
PacBio recommends to use size-selection beads are to remove short DNA fragments and enrich for the long fragments that are most valuable for downstream analysis. Although size-selection beads have many advantages, alternative methods — like gels and DNA precipitation — offer more flexibility in defining a size cutoff. This technical note provides guidance for alternative methods of size selection of WGS libraries prepared with the SMRTbell prep kit 3.0.
PacBio recommends that DNA shearing for whole genome sequencing be performed on the Diagenode Megaruptor 3 system, which is the only system validated by PacBio for shearing, in order to produce consistent HiFi yield and quality. When a Megaruptor 3 system is unavailable, the Covaris g-TUBE presents an alternative shearing method that does not require instrumentation beyond a standard microcentrifuge. This Technical note provides guidelines for using the Covaris g-TUBE for DNA shearing.
Sample preparation is a critical factor for whole genome sequencing (WGS) projects that impacts sequencing yield, read length, and ultimately performance for variant calling and genome assembly. Best practices to optimize performance for PacBio HiFi sequencing are to collect whole human blood, store with potassium EDTA as the anticoagulant for fewer than 2 days at 4°C, and extract DNA from 200 μL of blood using the Nanobind CBB Big DNA kit. When it is not possible to extract from fresh samples, storing samples at 4°C maximizes extraction yield and minimizes degradation for at least 9 days.
Technical note: Preparing samples for PacBio whole genome sequencing for de novo assembly – Collection and storage
Single Molecule, Real-Time (SMRT) Sequencing uses the natural process of DNA replication to sequence long fragments of native DNA. As such, starting with high-quality, high molecular weight (HMW) genomic DNA (gDNA) will result in better sequencing performance across difficult to sequence regions of the genome. To obtain the highest quality, long DNA it is important to start with sample types compatible with HMW DNA extraction methods. This technical note is intended to give general guidance on sample collection, preparation, and storage across a range of commonly encountered sample types used for SMRT Sequencing whole genome projects. It is important to note that all samples and projects are unique and may not be comprehensively addressed in this document.
Single Molecule, Real-Time (SMRT) Sequencing uses the natural process of DNA replication to sequence long fragments of native DNA in order to produce highly accurate long reads, or HiFi reads. As such, starting with high-quality, high molecular weight (HMW) genomic DNA (gDNA) will result in longer libraries and better performance during sequencing. This technical note is intended to give recommendations, tips and tricks for the extraction of DNA, as well as assessing and preserving the quality and size of your DNA sample to be used for HiFi sequencing.