June 1, 2021  |  

Harnessing kinetic information in Single-Molecule, Real-Time Sequencing.

Single-Molecule Real-Time (SMRT) DNA sequencing is unique in that nucleotide incorporation events are monitored in real time, leading to a wealth of kinetic information in addition to the extraction of the primary DNA sequence. The dynamics of the DNA polymerase that is observed adds an additional dimension of sequence-dependent information, and can be used to learn more about the molecule under study. First, the primary sequence itself can be determined more accurately. The kinetic data can be used to corroborate or overturn consensus calls and even enable calling bases in problematic sequence contexts. Second, using the kinetic information, we can detect and discriminate numerous chemical base modifications as a by-product of ordinary sequencing. Examples of applying these capabilities include (i) the characterization of the epigenome of microorganisms by directly sequencing the three common prokaryotic epigenetic base modifications of 4-methylcytosine, 5- methylcytosine and 6-methyladenine; (ii) the characterization of known and novel methyltransferase activities; (iii) the direct sequencing and differentiation of the four eukaryotic epigenetic forms of cytosine (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxylcytosine) with first applications to map them with single base-pair and DNA strand resolution across mammalian genomes; (iv) the direct sequencing and identification of numerous modified DNA bases arising from DNA damage; and (v) an exploration of the mitochondrial genome for known and novel base modifications. We will show our progress towards a generic, open-source algorithm for exploiting kinetic information for any of these purposes.


June 1, 2021  |  

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens.

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single-nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non- pathogenic to pathogenic states. Therefore, sequencing methods which provide complete, de novo genome assemblies and epigenomes are necessary to fully characterize infectious disease agents in an unbiased, hypothesis-free manner. Hybrid assembly methods have been described that combine long sequence reads from SMRT DNA Sequencing with short reads (SMRT CCS (circular consensus) or second-generation reads), wherein the short reads are used to error-correct the long reads which are then used for assembly. We have developed a new paradigm for microbial de novo assemblies in which SMRT sequencing reads from a single long insert library are used exclusively to close the genome through a hierarchical genome assembly process, thereby obviating the need for a second sample preparation, sequencing run, and data set. We have applied this method to achieve closed de novo genomes with accuracies exceeding QV50 (>99.999%) for numerous disease outbreak samples, including E. coli, Salmonella, Campylobacter, Listeria, Neisseria, and H. pylori. The kinetic information from the same SMRT Sequencing reads is utilized to determine epigenomes. Approximately 70% of all methyltransferase specificities we have determined to date represent previously unknown bacterial epigenetic signatures. With relatively short sequencing run times and automated analysis pipelines, it is possible to go from an unknown DNA sample to its complete de novo genome and epigenome in about a day.


June 1, 2021  |  

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non-pathogenic to pathogenic states. Therefore, sequencing methods which provide complete, de novo genome assemblies and epigenomes are necessary to fully characterize infectious disease agents in an unbiased, hypothesis-free manner. Hybrid assembly methods have been described that combine long sequence reads from SMRT DNA sequencing with short, high-accuracy reads (SMRT (circular consensus sequencing) CCS or second-generation reads) to generate long, highly accurate reads that are then used for assembly. We have developed a new paradigm for microbial de novo assemblies in which long SMRT sequencing reads (average readlengths >5,000 bases) are used exclusively to close the genome through a hierarchical genome assembly process, thereby obviating the need for a second sample preparation, sequencing run and data set. We have applied this method to achieve closed de novo genomes with accuracies exceeding QV50 (>99.999%) to numerous disease outbreak samples, including E. coli, Salmonella, Campylobacter, Listeria, Neisseria, and H. pylori. The kinetic information from the same SMRT sequencing reads is utilized to determine epigenomes. Approximately 70% of all methyltransferase specificities we have determined to date represent previously unknown bacterial epigenetic signatures. The process has been automated and requires less than 1 day from an unknown DNA sample to its complete de novo genome and epigenome.


June 1, 2021  |  

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark discoveries in biochemistry and genetics. We show that de novo assembly of a new strain yields complete assemblies of entire chromosomes, and additionally contains entire centromeric sequences. Base-modification analyses reveal candidate sites of increased interpulse duration (IPD) ratio, that may signify regions of 5mC, 5hmC, or 6mA base modifications. Iso-seq method provides full-length transcript evidence for comprehensive gene annotation, as well as context to the base-modifications in the newly assembled genome. Projects that integrate multiple genome-wide assays could become common practice for identifying genomic elements and understanding their function in new strains and organisms.


June 1, 2021  |  

Developments in PacBio metagenome sequencing: Shotgun whole genomes and full-length 16S.

The assembly of metagenomes is dramatically improved by the long read lengths of SMRT Sequencing. This is demonstrated in an experimental design to sequence a mock community from the Human Microbiome Project, and assemble the data using the hierarchical genome assembly process (HGAP) at Pacific Biosciences. Results of this analysis are promising, and display much improved contiguity in the assembly of the mock community as compared to publicly available short-read data sets and assemblies. Additionally, the use of base modification information to make further associations between contigs provides additional data to improve assemblies, and to distinguish between members within a microbial community. The epigenetic approach is a novel validation method unique to SMRT Sequencing. In addition to whole-genome shotgun sequencing, SMRT Sequencing also offers improved classification resolution and reliability of metagenomic and microbiome samples by the full-length sequencing of 16S rRNA (~1500 bases long). Microbial communities can be detected at the species level in some cases, rather than being limited to the genus taxonomic classification as constrained by short-read technologies. The performance of SMRT Sequencing for these metagenomic samples achieved >99% predicted concordance to reference sequences in cecum, soil, water, and mock control investigations for bacterial 16S. Community samples are estimated to contain from 2.3 and up to 15 times as many species with abundance levels as low as 0.05% compared to the identification of phyla groups.


June 1, 2021  |  

SMRT Sequencing solutions for investigative studies to understand evolutionary processes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers to understand molecular mechanisms in evolution and gain insight into adaptive strategies. With read lengths exceeding 10 kb, we are able to sequence high-quality, closed microbial genomes with associated plasmids, and investigate large genome complexities, such as long, highly repetitive, low-complexity regions and multiple tandem-duplication events. Improved genome quality, observed at 99.9999% (QV60) consensus accuracy, and significant reduction of gap regions in reference genomes (up to and beyond 50%) allow researchers to better understand coding sequences with high confidence, investigate potential regulatory mechanisms in noncoding regions, and make inferences about evolutionary strategies that are otherwise missed by the coverage biases associated with short- read sequencing technologies. Additional benefits afforded by SMRT Sequencing include the simultaneous capability to detect epigenomic modifications and obtain full-length cDNA transcripts that obsolete the need for assembly. With direct sequencing of DNA in real-time, this has resulted in the identification of numerous base modifications and motifs, which genome-wide profiles have linked to specific methyltransferase activities. Our new offering, the Iso-Seq Application, allows for the accurate differentiation between transcript isoforms that are difficult to resolve with short-read technologies. PacBio reads easily span transcripts such that both 5’/3’ primers for cDNA library generation and the poly-A tail are observed. As such, exon configuration and intron retention events can be analyzed without ambiguity. This technological advance is useful for characterizing transcript diversity and improving gene structure annotations in reference genomes. We review solutions available with SMRT Sequencing, from targeted sequencing efforts to obtaining reference genomes (>100 Mb). This includes strategies for identifying microsatellites and conducting phylogenetic comparisons with targeted gene families. We highlight how to best leverage our long reads that have exceeded 20 kb in length for research investigations, as well as currently available bioinformatics strategies for analysis. Benefits for these applications are further realized with consistent use of size selection of input sample using the BluePippin™ device from Sage Science as demonstrated in our genome improvement projects. Using the latest P5-C3 chemistry on model organisms, these efforts have yielded an observed contig N50 of ~6 Mb, with the longest contig exceeding 12.5 Mb and an average base quality of QV50.


June 1, 2021  |  

Whole genome sequencing and epigenome characterization of cancer cells using the PacBio platform.

The comprehensive characterization of cancer genomes and epigenomes for understanding drug resistance remains an important challenge in the field of oncology. For example, PC-9, a non-small cell lung cancer (NSCL) cell line, contains a deletion mutation in exon 19 (DelE746A750) of EGRF that renders it sensitive to erlotinib, an EGFR inhibitor. However, sustained treatment of these cells with erlotinib leads to drug-tolerant cell populations that grow in the presence of erlotinib. However, the resistant cells can be resensitized to erlotinib upon treatment with methyltransferase inhibitors, suggesting a role of epigenetic modification in development of drug resistance. We have characterized for the first time cancer genomes of both drug-sensitive and drug-resistant PC- 9 cells using long-read PacBio sequencing. The PacBio data allowed us to generate a high-quality, de novo assembly of this cancer genome, enabling the detection of forms of genomic variations at all size scales, including SNPs, structural variations, copy number alterations, gene fusions, and translocations. The data simultaneously provide a global view of epigenetic DNA modifications such as methylation. We will present findings on large-scale changes in the methylation status across the cancer genome as a function of drug sensitivity.


June 1, 2021  |  

Epigenome characterization of human genomes using the PacBio platform

In addition to the genome and transcriptome, epigenetic information is essential to understand biological processes and their regulation, and their misregulation underlying disease. Traditionally, epigenetic DNA modifications are detected using upfront sample preparation steps such as bisulfite conversion, followed by sequencing. Bisulfite sequencing has provided a wealth of knowledge about human epigenetics, however it does not access the entire genome due to limitations in read length and GC- bias of the sequencing technologies used. In contrast, Single Molecule, Real-Time (SMRT) DNA Sequencing is unique in that it can detect DNA base modifications as part of the sequencing process. It can thereby leverage the long read lengths and lack of GC bias for more comprehensive views of the human epigenome. I will highlight several examples of this capability towards the generation of new biological insights, including the resolution of methylation states in repetitive and GC-rich regions of the genome, and large-scale changes in the methylation status across a cancer genome as a function of drug sensitivity.


June 1, 2021  |  

A comprehensive study of the sugar pine (Pinus lambertiana) transcriptome implemented through diverse next-generation sequencing approaches

The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46 SMRT cells) reads, 33,720 unigenes were de novo assembled. Comparison of sequencing technologies revealed improved coverage with Illumina HiSeq reads and better splice variant detection with PacBio Iso-Seq reads. The genes identified as unique to each library ranges from 199 transcripts (basket seedling) to 3,482 transcripts (female cones). In total, 10,026 transcripts were shared by all libraries. Genes differentially expressed in response to these provided insight on abiotic and biotic stress responses. To analyze orthologous sequences, we compared the translated sequences against 19 plant species, identifying 7,229 transcripts that clustered uniquely among the conifers. We have generated here a high quality transcriptome from one WPBR susceptible and one WPBR resistant sugar pine individual. Through the comprehensive tissue sampling and the depth of the sequencing achieved, detailed information on disease resistance can be further examined.


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  |  

Enrichment of unamplified DNA and long-read SMRT Sequencing in unlocking the underlying biological disease mechanisms of repeat expansion disorders

For many of the repeat expansion disorders, the disease gene has been discovered, however the underlying biological mechanisms have not yet been fully understood. This is mainly due to technological limitations that do not allow for the needed base-pair resolution of the long, repetitive genomic regions. We have developed a novel, amplification-free enrichment technique that uses the CRISPR/Cas9 system to target large repeat expansions. This method, in conjunction with PacBio’s long reads and uniform coverage, enables sequencing of these complex genomic regions. By using a PCR-free amplification method, we are able to access not only the repetitive elements and interruption sequences accurately, but also the epigenetic information.


June 1, 2021  |  

Candidate gene screening using long-read sequencing

We have developed several candidate gene screening applications for both Neuromuscular and Neurological disorders. The power behind these applications comes from the use of long-read sequencing. It allows us to access previously unresolvable and even unsequencable genomic regions. SMRT Sequencing offers uniform coverage, a lack of sequence context bias, and very high accuracy. In addition, it is also possible to directly detect epigenetic signatures and characterize full-length gene transcripts through assembly-free isoform sequencing. In addition to calling the bases, SMRT Sequencing uses the kinetic information from each nucleotide to distinguish between modified and native bases.


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  |  

Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies

Target enrichment capture methods allow scientists to rapidly interrogate important genomic regions of interest for variant discovery, including SNPs, gene isoforms, and structural variation. Custom targeted sequencing panels are important for characterizing heterogeneous, complex diseases and uncovering the genetic basis of inherited traits with more uniform coverage when compared to PCR-based strategies. With the increasing availability of high-quality reference genomes, customized gene panels are readily designed with high specificity to capture genomic regions of interest, thus enabling scientists to expand their research scope from a single individual to larger cohort studies or population-wide investigations. Coupled with PacBio® long-read sequencing, these technologies can capture 5 kb fragments of genomic DNA (gDNA), which are useful for interrogating intronic, exonic, and regulatory regions, characterizing complex structural variations, distinguishing between gene duplications and pseudogenes, and interpreting variant haplotyes. In addition, SMRT® Sequencing offers the lowest GC-bias and can sequence through repetitive regions. We demonstrate the additional insights possible by using in-depth long read capture sequencing for key immunology, drug metabolizing, and disease causing genes such as HLA, filaggrin, and cancer associated genes.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.