Starting a sequencing project can be daunting. First of all, there are several types of sequencing technologies, each based on unique processes. At PacBio, we use a technology called Single Molecule, Real-Time (SMRT) sequencing.
Learn how SMRT sequencing works in this short video:
Although each sequencing project is unique, there are five main steps to go from DNA to discovery with SMRT sequencing:
Step 1: sample prep
Similar to cooking, for the best results, start with the best ingredients. The ideal sequencing starter is high molecular weight DNA. There are plenty of kits on the market that can help with this, and PacBio also maintains a site of DNA extraction protocols to aid these efforts.
Expert sample wrangler Olga Pettersson (@OlgaVPettersson) of SciLifeLab at Uppsala University, also advises: “Aim for getting molecules as long as you can, as pure as you can, as fresh as you can.”
When in doubt, you can always outsource the task to experts at other labs or sequencing centers, such as our certified service providers.
Step 2: library prep
Library preparation for all of the major next generation sequencing (NGS) platforms requires the ligation of specific adapter oligos to fragments of the DNA to be sequenced. The DNA has to be fragmented to the optimal length determined by the sequencing technology you are using.
PacBio uses a SMRTbell library format, in which DNA fragments are capped on both sides with ligated hairpin adapters, where the sequencing primers attach. This creates a circular template for the polymerase to navigate. These can be created for libraries of varying insert lengths — from 250 bp to greater than 25,000 bp. Samples can also be barcoded and multiplexed to increase throughput. Learn more about kits for fast and easy library preparation.
Step 3: sequencing
Once your library is prepared, the PacBio sequencing system, such as the Sequel II system, takes over. At the heart of SMRT sequencing is the SMRT Cell, which contains millions of tiny wells called zero-mode waveguides (ZMWs). Single molecules of DNA are immobilized in these wells, and as the polymerase incorporates each nucleotide, light is emitted, and nucleotide incorporation is measured in real time. The reactions are recorded in a format that can then be analyzed using on-instrument tools, as well as additional software.
You can explore this interactive to learn about the Sequel II system and SMRT sequencing applications including whole genome sequencing for de novo assembly, comprehensive variant detection, full-length RNA sequencing, metagenomic sequencing, and more.
With the current sequencing performance, a single SMRT Cell can be used to generate a reference-quality assembly of a 2 Gb genome, characterize a whole transcriptome and identify alternative splicing, or determine the composition of up to 96 microbiome samples. Find out what else you can do with a single SMRT Cell.
Step 4: data analysis
As mentioned, initial analysis occurs within the instrument itself to provide the sequence output. Secondary analysis can then be performed with the SMRT Link user interface and SMRT Analysis, which feature a suite of analytical applications and visualization tools.
Unique to SMRT sequencing is the ability to sequence the same DNA molecule multiple times generating highly accurate long reads, or HiFi reads. For users, like Jeremy Schmutz of the HudsonAlpha Institute of Biotechnology, this is quite beneficial.
“With HiFi reads, we can take the reads and do something with them right away. We don’t have to go through an enormous amount of downstream computation and processing to get to the point of having some sequence that we can evaluate,” he stated.
Depending on what you want to do with your data, there are also a range of tertiary analysis tools you can use, many of which have been developed by users. You can delve into example datasets to get to know SMRT sequencing data and check out PacBio DevNet for easy access to open-source community-developed analysis tools and other resources to help further your analysis and data interpretation.
Step 5: understanding biology
The final and most exciting part of all good sequencing projects is taking your study from data to discovery by using it to improve understanding of biology. Members of the SMRT community have already greatly expanded knowledge across a huge range of fields. Check out our blog and SMRT resources library, to find many examples of how SMRT sequencing has been applied to biological questions ranging from confirming the causative variant of a disease, to sex determination of the asparagus plant, or determining what microbes are best for cheese making.
For an additional introduction to SMRT sequencing, including a Q&A with expert users, check out this video.
If you are interested in using SMRT sequencing in your research and would like a free project consultation, connect with a PacBio scientist
Explore other posts in the Sequencing 101 series:
The evolution of DNA sequencing tools
Introduction to PacBio sequencing and the Sequel II system
Why are long reads important for studying viral genomes?
Looking beyond the single reference genome to a pangenome for every species
Understanding accuracy in DNA sequencing
The value of sequencing full-length RNA transcripts of DNA transcripts
Ploidy, haplotypes, and phasing — how to get more from your sequencing data
DNA extraction — tips, kits, and protocols
Video: Sequencing 101: how long-read sequencing improves access to genetic information