June 1, 2021  |  

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis

Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. The resulting library can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes using the SMRT Portal assembly program HGAP, plus base modification analysis. The entire process can be done in less than 3 days by standard laboratory personnel. This approach is particularly important for analysis of metagenomic communities, in which genomic DNA is often limited. From these samples, full-length 16S amplicons can be generated, prepped with the standard SMRTbell library prep protocol, and sequenced. Alternatively, a 2 kb sheared library, made from a few ng of input DNA, can also be used to elucidate the microbial composition of a community, and may provide information about biochemical pathways present in the sample. In both these cases, 1-2 kb reads with >99.9% accuracy can be obtained from Circular Consensus Sequencing.


June 1, 2021  |  

Multiplexing strategies for microbial whole genome SMRT Sequencing

The increased throughput of the RS II and Sequel Systems enables multiple microbes to be sequenced on a single SMRT Cell. This multiplexing can be readily achieved by simply incorporating a unique barcode for each microbe into the SMRTbell adapters after shearing genomic DNA using a streamlined library construction process. Incorporating a barcode without the requirement for PCR amplification prevents the loss of epigenetic information (e.g., methylation signatures), and the generation of chimeric sequences, while the modified protocol eliminates the need to build several individual SMRTbell libraries. We multiplexed up to 8 unique strains of H. pylori. Each strain was sheared, and processed through adapter ligation in a single, addition only reaction. The barcoded strains were then pooled in equimolar quantities, and processed through the remainder of the library preparation and purification steps. We demonstrate successful de novo microbial assembly and epigenetic analysis from all multiplexes (2 through 8-plex) using standard tools within SMRT Link Analysis using data generated from a single SMRTbell library, run on a single SMRT Cell. This process facilitates the sequencing of multiple microbial genomes in a single day, greatly increasing throughput and reducing costs per genome assembly.


June 1, 2021  |  

Highly contiguous de novo human genome assembly and long-range haplotype phasing using SMRT Sequencing

The long reads, random error, and unbiased sampling of SMRT Sequencing enables high quality, de novo assembly of the human genome. PacBio long reads are capable of resolving genomic variations at all size scales, including SNPs, insertions, deletions, inversions, translocations, and repeat expansions, all of which are both important in understanding the genetic basis for human disease, and difficult to access via other technologies. In demonstration of this, we report a new high-quality, diploid-aware de novo assembly of Craig Venter’s well-studied genome.


June 1, 2021  |  

Multiplexing strategies for microbial whole genome SMRT Sequencing

As the throughput of the PacBio Systems continues to increase, so has the desire to fully utilize SMRT Cell sequencing capacity to multiplex microbes for whole genome sequencing. Multiplexing is readily achieved by incorporating a unique barcode for each microbe into the SMRTbell adapters and using a streamlined library preparation process. Incorporating barcodes without PCR amplification prevents the loss of epigenetic information and the generation of chimeric sequences, while eliminating the need to generate separate SMRTbell libraries. We multiplexed the genomes of up to 8 unique strains of H. pylori. Each genome was sheared and processed through adapter ligation in a single, addition-only reaction. The barcoded samples were pooled in equimolar quantities and a single SMRTbell library was prepared. We demonstrate successful de novo microbial assembly from all multiplexes tested (2- through 8-plex) using data generated from a single SMRTbell library, run on a single SMRT Cell with the PacBio RS II, and analyzed with standard SMRT Analysis assembly methods. This strategy was successful using both small (1.6 Mb, H. pylori) and medium (5 Mb, E. coli) genomes. This protocol facilitates the sequencing of multiple microbial genomes in a single run, greatly increasing throughput and reducing costs per genome.


June 1, 2021  |  

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome using long-read sequencing

Sequence-based estimation of genetic diversity of Plasmodium falciparum, the most lethal malarial parasite, has proved challenging due to a lack of a complete genomic assembly. The skewed AT-richness (~80.6% (A+T)) of its genome and the lack of technology to assemble highly polymorphic sub-telomeric regions that contain clonally variant, multigene virulence families (i.e. var and rifin) have confounded attempts using short-read NGS technologies. Using single molecule, real-time (SMRT) sequencing, we successfully compiled all 14 nuclear chromosomes of the P. falciparum genome from telomere-to-telomere in single contigs. Specifically, amplification-free sequencing generated reads of average length 12 kb, with =50% of the reads between 15.5 and 50 kb in length. A hierarchical genome assembly process (HGAP), was used to assemble the P. falciparum genome de novo. This assembly accurately resolved centromeres (~90-99% (A+T)) and sub-telomeric regions, and identified large insertions and duplications in the genome that added extra genes to the var and rifin virulence families, along with smaller structural variants such as homopolymer tract expansions. These regions can be used as markers for genetic diversity during comparative genome analyses. Moreover, identifying the polymorphic and repetitive sub-telomeric sequences of parasite populations from endemic areas might inform the link between structural variation and phenotypes such as virulence, drug resistance and disease transmission.


June 1, 2021  |  

Multiplexed complete microbial genomes on the Sequel System

Microbes play an important role in nearly every part of our world, as they affect human health, our environment, agriculture, and aid in waste management. Complete closed genome sequences, which have become the gold standard with PacBio long-read sequencing, can be key to understanding microbial functional characteristics. However, input requirements, consumables costs, and the labor required to prepare and sequence a microbial genome have in the past put PacBio sequencing out of reach for some larger projects. We have developed a multiplexed library prep approach that is simple, fast, and cost-effective, and can produce 4 to 16 closed bacterial genomes from one Sequel SMRT Cell. Additionally, we are introducing a streamlined analysis pipeline for processing multiplexed genome sequence data through de novo HGAP assembly, making the entire process easy for lab personnel to perform. Here we present the entire workflow from shearing through assembly, with times for each step. We show HGAP assembly results with single or very few contigs from bacteria from different size genomes, sequenced without or with size selection. These data illustrate the benefits and potential of the PacBio multiplexed library prep and the Sequel System for sequencing large numbers of microbial genomes.


June 1, 2021  |  

Best practices for whole genome sequencing using the Sequel System

Plant and animal whole genome sequencing has proven to be challenging, particularly due to genome size, high density of repetitive elements and heterozygosity. The Sequel System delivers long reads, high consensus accuracy and uniform coverage, enabling more complete, accurate, and contiguous assemblies of these large complex genomes. The latest Sequel chemistry increases yield up to 8 Gb per SMRT Cell for long insert libraries >20 kb and up to 10 Gb per SMRT Cell for libraries >40 kb. In addition, the recently released SMRTbell Express Template Prep Kit reduces the time (~3 hours) and DNA input (~3 µg), making the workflow easy to use for multi- SMRT Cell projects. Here, we recommend the best practices for whole genome sequencing and de novo assembly of complex plant and animal genomes. Guidelines for constructing large-insert SMRTbell libraries (>30 kb) to generate optimal read lengths and yields using the latest Sequel chemistry are presented. We also describe ways to maximize library yield per preparation from as littles as 3 µg of sheared genomic DNA. The combination of these advances makes plant and animal whole genome sequencing a practical application of the Sequel System.


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