June 1, 2021  |  

Advances in sequence consensus and clustering algorithms for effective de novo assembly and haplotyping applications.

One of the major applications of DNA sequencing technology is to bring together information that is distant in sequence space so that understanding genome structure and function becomes easier on a large scale. The Single Molecule Real Time (SMRT) Sequencing platform provides direct sequencing data that can span several thousand bases to tens of thousands of bases in a high-throughput fashion. In contrast to solving genomic puzzles by patching together smaller piece of information, long sequence reads can decrease potential computation complexity by reducing combinatorial factors significantly. We demonstrate algorithmic approaches to construct accurate consensus when the differences between reads are dominated by insertions and deletions. High-performance implementations of such algorithms allow more efficient de novo assembly with a pre-assembly step that generates highly accurate, consensus-based reads which can be used as input for existing genome assemblers. In contrast to recent hybrid assembly approach, only a single ~10 kb or longer SMRTbell library is necessary for the hierarchical genome assembly process (HGAP). Meanwhile, with a sensitive read-clustering algorithm with the consensus algorithms, one is able to discern haplotypes that differ by less than 1% different from each other over a large region. One of the related applications is to generate accurate haplotype sequences for HLA loci. Long sequence reads that can cover the whole 3 kb to 4 kb diploid genomic regions will simplify the haplotyping process. These algorithms can also be applied to resolve individual populations within mixed pools of DNA molecules that are similar to each, e.g., by sequencing viral quasi-species samples.


June 1, 2021  |  

SMRT Sequencing solutions for large genomes and transcriptomes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers in large genome complexities, such as long, highly repetitive, low-complexity regions and duplication events, and differentiating between transcript isoforms that are difficult to resolve with short-read technologies. We present solutions available for both reference genome improvement (>100 MB) and transcriptome research to best leverage long reads that have exceeded 20 Kb in length. Benefits for these applications are further realized with consistent use of size-selection of input sample using the BluePippin™ device from Sage Science. Highlights from our genome assembly projects using the latest P5-C3 chemistry on model organisms will be shared. Assembly contig N50 have exceeded 6 Mb and we observed longest contig exceeding 12.5 Mb with an average base quality of QV50. Additionally, the value of long, intact reads to provide a no-assembly approach to investigate transcript isoforms using our Iso-Seq Application will be presented.


June 1, 2021  |  

Complete resequencing of extended genomic regions using fosmid target capture and single molecule real-time (SMRT) long read sequencing technology.

A longstanding goal of genomic analysis is the identification of causal genetic factors contributing to disease. While the common disease/common variant hypothesis has been tested in many genome-wide association studies, few advancements in identifying causal variation have been realized, and instead recent findings point away from common variants towards aggregate rare variants as causal. A challenge is obtaining complete phased genomic sequences over extended genomic regions from sufficient numbers of cases and controls to identify all potential variation causal of a disease. To address this, we modified methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generaton that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As proof of principal, we validated the approach by resequencing four 800 kbp segments that span a major histocompatibility complex (MHC) common extended haplotype (CEH) associated with disease. The data revealed the extent of conservation exposing a near identity among four DR4 CEHs over conserved regions, detailing rare variation and measuring sequence accuracy. In a second test, we sequenced the complete KIR haplotypes from 8 individuals within a specific timeframe and cost. Single molecule long-read sequencing technology generated contiguous full-­length fosmid sequences of 30 to 40 kb in a single read, allowing assembly of resolved haplotypes with very little data processing. All of the sequences produced from these projects were contiguous, phased, with accuracy above 99.99%. The results demonstrated that cost-effective scale-­up is possible to generate scores to hundreds of phased chromosomal sequences of extended lengths that can encompass genomic regions associated with disease.


June 1, 2021  |  

Barcoding strategies for multiplexing of samples using a long-read sequencing technology.

We have developed barcoding reagents and workflows for multiplexing amplicons or fragmented native genomic (DNA) prior to Single Molecule, Real-Time (SMRT) Sequencing. The long reads of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases (kb) of sequence. This feature is particularly useful in the context of mutational analysis or SNP confirmation, where a large number of samples are generated routinely. To validate this workflow, a set of 384 1.7-kb amplicons, each derived from variants of the Phi29 DNA polymerase gene, were barcoded during amplification, pooled, and sequenced on a single SMRT Cell. To demonstrate the applicability of the method to longer inserts, a library of 96 5-kb clones derived from the E. coli genome was sequenced.


June 1, 2021  |  

Progress Toward a Low Budget Reference Grade Genome Assembly

Reference quality de novo genome assemblies were once solely the domain of large, well-funded genome projects. While next-generation short read technology removed some of the cost barriers, accurate chromosome-scale assembly remains a real challenge. Here we present efforts to de novo assemble the goat (Capra hircus) genome. Through the combination of single-molecule technologies from Pacific Biosciences (sequencing) and BioNano Genomics (optical mapping) coupled with high-throughput chromosome conformation capture sequencing (Hi-C), an inbred San Clemente goat genome has been sequenced and assembled to a high degree of completeness at a relatively modest cost. Starting with 38 million PacBio reads, we integrated the MinHash Alignment Process (MHAP) with the Celera Assembler (CA) to produce an assembly composed of 3110 contigs with a contig N50 size of 4.7 Mb. This assembly was scaffolded with BioNano genome maps derived from a single IrysChip into 333 scaffolds with an N50 of 23.1 Mb including the complete scaffolding of chromosome 20. Finally, cis-chromosome associations were determined by Hi-C, yielding complete reconstruction of all autosomes into single scaffolds with a final N50 of 91.7 Mb. We hope to demonstrate that our methods are not only cost effective, but improve our ability to annotate challenging genomic regions such as highly repetitive immune gene clusters.


June 1, 2021  |  

Long read sequencing technology to solve complex genomic regions assembly in plants

Numerous whole genome sequencing projects already achieved or ongoing have highlighted the fact that obtaining a high quality genome sequence is necessary to address comparative genomics questions such as structural variations among genotypes and gain or loss of specific function. Despite the spectacular progress that has been done regarding sequencing technologies, accurate and reliable data are still challenging, at the whole genome scale but also when targeting specific genomic regions. These issues are even more noticeable for complex plant genomes. Most plant genomes are known to be particularly challenging due to their size, high density of repetitive elements and various levels of ploidy. To overcome these issues, we have developed a strategy in order to reduce the genome complexity by using the large insert BAC libraries combined with next generation sequencing technologies. We have compared two different technologies (Roche-454 and Pacific Biosciences PacBio RS II) to sequence pools of BAC clones in order to obtain the best quality sequence. We targeted nine BAC clones from different species (maize, wheat, strawberry, barley, sugarcane and sunflower) known to be complex in terms of sequence assembly. We sequenced the pools of the nine BAC clones with both technologies. We have compared results of assembly and highlighted differences due to the sequencing technologies used. We demonstrated that the long reads obtained with the PacBio RS II technology enables to obtain a better and more reliable assembly notably by preventing errors due to duplicated or repetitive sequences in the same region.


June 1, 2021  |  

Un-zipping diploid genomes – revealing all kinds of heterozygous variants from comprehensive hapltotig assemblies

Outside of the simplest cases (haploid, bacteria, or inbreds), genomic information is not carried in a single reference per individual, but rather has higher ploidy (n=>2) for almost all organisms. The existence of two or more highly related sequences within an individual makes it extremely difficult to build high quality, highly contiguous genome assemblies from short DNA fragments. Based on the earlier work on a polyploidy aware assembler, FALCON (https://github.com/PacificBiosciences/FALCON), we developed new algorithms and software (FALCON-unzip) for de novo haplotype reconstructions from SMRT Sequencing data. We apply the algorithms and the prototype software for (1) a highly repetitive diploid fungal genome (Clavicorona pyxidata) and (2) an F1 hybrid from two inbred Arabidopsis strains: CVI-0 and COL-0. For the fungal genome, we achieved an N50 of 1.53 Mb (of the 1n assembly contigs) of the ~42 Mb 1n genome and an N50 of the haplotigs of 872 kb from a 95X read length N50 ~16 kb dataset. We found that ~ 45% of the genome was highly heterozygous and ~55% of the genome was highly homozygous. We developed methods to assess the base-level accuracy and local haplotype phasing accuracy of the assembly with short-read data from the Illumina platform. For the Arabidopsis F1 hybrid genome, we found that 80% of the genome could be separated into haplotigs. The long range accuracy of phasing haplotigs was evaluated by comparing them to the assemblies from the two inbred parental lines. We show that a more complete view of all haplotypes could provide useful biological insights through improved annotation, characterization of heterozygous variants of all sizes, and resolution of differential allele expression. Finally, we applied this method to WGS human data sets to demonstrate the potential for resolving complicated, medically-relevant genomic regions.


June 1, 2021  |  

Immune regions are no longer incomprehensible with SMRT Sequencing

The complex immune regions of the genome, including MHC and KIR, contain large copy number variants (CNVs), a high density of genes, hyper-polymorphic gene alleles, and conserved extended haplotypes (CEH) with enormous linkage disequilibrium (LDs). This level of complexity and inherent biases of short-read sequencing make it challenging for extracting immune region haplotype information from reference-reliant, shotgun sequencing and GWAS methods. As NGS based genome and exome sequencing and SNP arrays have become a routine for population studies, numerous efforts are being made for developing software to extract and or impute the immune gene information from these datasets. Despite these efforts, the fine mapping of causal variants of immune genes for their well-documented association with cancer, drug-induced hypersensitivity and immune-related diseases, has been slower than expected. This has in many ways limited our understanding of the mechanisms leading to immune disease. In the present work, we demonstrate the advantages of long reads delivered by SMRT Sequencing for assembling complete haplotypes of MHC and KIR gene clusters, as well as calling correct genotypes of genes comprised within them. All the genotype information is detected at allele- level with full phasing information across SNP-poor regions. Genotypes were called correctly from targeted gene amplicons, haplotypes, as well as from a completely assembled 5 Mb contig of the MHC region from a de novo assembly of whole genome shotgun data. De novo analysis pipeline used in all these approaches allowed for reference-free analysis without imputation, a key for interrogation without prior knowledge about ethnic backgrounds. These methods are thus easily adoptable for previously uncharacterized human or non-human species.


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  |  

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.


June 1, 2021  |  

Characterization of the Poly-T variants in the TOMM40 gene using PacBio long reads

Genes associated with several neurological disorders have been shown to be highly polymorphic. Targeted sequencing of these genes using NGS technologies is a powerful way to increase the cost-effectiveness of variant discovery and detection. However, for a comprehensive view of these target genes, it is necessary to have complete and uniform coverage across regions of interest. Unfortunately, short-read sequencing technologies are not ideal for these types of studies as they are prone to mis-mapping and often fail to span repetitive regions. Targeted sequencing with PacBio long reads provides the unique advantage of single-molecule observations of complex genomic regions. PacBio long reads not only provide continuous sequence data though polymorphic or repetitive regions, but also have no GC bias. Here we describe the characterization of the poly-T locus in TOMM40, a gene known to be associated with progression to Alzheimer’s, using PacBio long reads. Probes were designed to capture a 20 kb region comprising the TOMM40 and ApoE genes. Target regions were captured in multiple cell lines and sequencing libraries made using standard sample preparation methods. We will present our results on the poly-T structural variants that we observed in TOMM40 in these cell lines. We will also present our results on probe design optimization and barcoding strategies for a cost-effective solution.


June 1, 2021  |  

Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders

Nucleotide repeat expansions are a major cause of neurological and neuromuscular disease in humans, however, the nature of these genomic regions makes characterizing them extremely challenging. Accurate DNA sequencing of repeat expansions using short-read sequencing technologies is difficult, as short-read technologies often cannot read through regions of low sequence complexity. Additionally, these short reads do not span the entire region of interest and therefore sequence assembly is required. Lastly, most target enrichment methods are reliant upon amplification which adds the additional caveat of PCR bias. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific targeting of individual human genes. This method, in conjunction with PacBio’s long reads and uniform coverage, enables sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples and this strategy, we have successfully targeted the loci of Huntington’s Disease (HTT; CAG repeat), Fragile X (FMR1; CGG repeat), ALS (C9orf72; GGGGCC repeat), and Spinocerebellar ataxia type 10 (SCA10; variable ATTCT repeat) for examination. With this data, we demonstrate the ability to isolate hundreds of individual on-target molecules in a single SMRT Cell and accurately sequence through long repeat stretches, regardless of the extreme GC-content. The method is compatible with multiplexing of multiple targets and multiple samples in a single reaction. This technique also captures native DNA molecules for sequencing, allowing for the possibility of direct detection and characterization of epigenetic signatures.


June 1, 2021  |  

Target enrichment using a neurology panel for 12 barcoded genomic DNA samples on the PacBio SMRT Sequencing platform

Target enrichment is a powerful tool for studies involved in understanding polymorphic SNPs with phasing, tandem repeats, and structural variations. With increasing availability of reference genomes, researchers can easily design a cost-effective targeted investigation with custom probes specific to regions of interest. Using PacBio long-read technology in conjunction with probe capture, we were able to sequence multi-kilobase enriched regions to fully investigate intronic and exonic regions, distinguish haplotypes, and characterize structural variations. Furthermore, we demonstrate this approach is advantageous for studying complex genomic regions previously inaccessible through other sequencing platforms. In the present work, 12 barcoded genomic DNA (gDNA) samples were sheared to 6 kb for target enrichment analysis using the Neurology panel provided by Roche NimbleGen. Probe-captured DNA was used to make SMRTbell libraries for SMRT Sequencing on the PacBio RS II. Our results demonstrate the ability to multiplex 12 samples and achieve 1300x enrichment of targeted regions. In addition, we achieved an even representation of on-target rate of 70% across the 12 barcoded genomic DNA samples.


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