June 1, 2021  |  

A comparison of 454 GS FLX Ti and PacBio RS in the context of characterizing HIV-1 intra-host diversity.

PacBio 2013 User Group Meeting Presentation Slides: Lance Hepler from UC San Diego’s Center for AIDS Research used the PacBio RS to study intra-host diversity in HIV-1. He compared PacBio’s performance to that of 454® sequencer, the platform he and his team previously used. Hepler noted that in general, there was strong agreement between the platforms; where results differed, he said that PacBio data had significantly better reproducibility and accuracy. “PacBio does not suffer from local coverage loss post-processing, whereas 454 has homopolymer problems,” he noted. Hepler said they are moving away from using 454 in favor of the PacBio system.


June 1, 2021  |  

Resolving the ‘dark matter’ in genomes.

Second-generation sequencing has brought about tremendous insights into the genetic underpinnings of biology. However, there are many functionally important and medically relevant regions of genomes that are currently difficult or impossible to sequence, resulting in incomplete and fragmented views of genomes. Two main causes are (i) limitations to read DNA of extreme sequence content (GC-rich or AT-rich regions, low complexity sequence contexts) and (ii) insufficient read lengths which leave various forms of structural variation unresolved and result in mapping ambiguities.


June 1, 2021  |  

Assembly of complete KIR haplotypes from a diploid individual by the direct sequencing of full-length fosmids.

We show that linearizing and directly sequencing full-length fosmids simplifies the assembly problem such that it is possible to unambiguously assemble individual haplotypes for the highly repetitive 100-200 kb killer Ig-like receptor (KIR) gene loci of chromosome 19. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the KIR locus, means that sequence assembly of pooled samples into complete haplotypes is difficult and in many cases impossible. The current maximum read length generated by SMRT Sequencing exceeds the length of a 40 kb fosmid; it is therefore possible to span an entire fosmid in one sequencing read. Shearing, sequencing and assembling fosmids in a shotgun approach is prone to errors when the underlying sequence is highly repetitive. We show that it is possible to directly sequence linearized fosmids and generate a high-quality consensus by simple alignment, removing the need for an error-prone assembly step. The high-quality sequence of complete fosmids can then be tiled into full haplotypes. We demonstrate the method on DNA samples from a number of individuals and fully recover the sequence of both haplotypes from a pool of KIR fosmids. The ability to haplotype and sequence complex immunogenetic regions will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. This simple and robust approach can be scaled-up allowing a complex genomic region to be sequenced at a population level. We expect such sequencing to be valuable in disease association research.


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  |  

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  |  

Screening and characterization of causative structural variants for bipolar disorder in a significantly linked chromosomal region onXq24-q27 in an extended pedigree from a genetic isolate

Bipolar disorder (BD) is a phenotypically and genetically complex and debilitating neurological disorder that affects 1% of the worldwide population. There is compelling evidence from family, twin and adoption studies supporting the involvement of a genetic predisposition in BD with estimated heritability up to ~ 80%. The risk in first-degree relatives is ten times higher than in the general population. Linkage and association studies have implicated multiple putative chromosomal loci for BP susceptibility, however no disease genes have been identified to date.


June 1, 2021  |  

Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease. 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 SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows the sequencing of complex genomic regions that cannot be investigated with other technologies.


June 1, 2021  |  

Targeted enrichment without amplification and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease. 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 SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows the 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 a number of repeat expansion disorders (HTT, FMR1, ATXN10, C9orf72). With this data, we demonstrate the ability to isolate hundreds of individual on-target molecules and accurately sequence through long repeat stretches, regardless of the extreme GC-content, followed by accurate sequencing on a single PacBio RS II SMRT Cell or Sequel SMRT Cell 1M. The method is compatible with multiplexing of multiple targets and multiple samples in a single reaction. Furthermore, this technique also preserves native DNA molecules for sequencing, allowing for the possibility of direct detection and characterization of epigenetic signatures. We demonstrate detection of 5-mC in human promoter sequences and CpG islands.


June 1, 2021  |  

Best practices for diploid assembly of complex genomes using PacBio: A case study of Cascade Hops

A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over earlier short-read assemblies. The genome is primarily assembled as haplotypes due to the outbred nature of the organism. We explore patterns of haplotype divergence across the assembly and present strategies to deduplicate haplotypes prior to scaffolding


June 1, 2021  |  

Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease.


June 1, 2021  |  

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be economical for obtaining sequence information for defined regions of the genome. However, most target enrichment methods are reliant upon some form of amplification which can negatively impact downstream analysis. For example, amplification removes epigenetic marks present in native DNA, including nucleotide methylation, which are hypothesized to contribute to disease mechanisms in some disorders. In addition, some genomic regions known to be causative of many genetic disorders have extreme GC content and/or repetitive sequences that tend to be recalcitrant to faithful amplification. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system to target individual genes. This method, in conjunction with the long reads, high consensus accuracy, and uniform coverage of SMRT Sequencing, allows accurate sequence analysis of complex genomic regions that cannot be investigated with other technologies. Using this strategy, we have successfully targeted a number of repeat expansion disorder loci (HTT, FMR1, ATXN10, C9orf72).With this data, we demonstrate the ability to isolate thousands of individual on-target molecules and, using the Sequel System, accurately sequence through long repeats regardless of the extreme GC-content. The method is compatible with multiplexing of multiple target loci and multiple samples in a single reaction. Furthermore, because there is no amplification step, this technique also preserves native DNA molecules for sequencing, allowing for the direct detection and characterization of epigenetic signatures. To this end, we demonstrate the detection of 5-mC in the CGG repeat of the FMR1 gene that is responsible for Fragile X syndrome.


June 1, 2021  |  

TLA & long-read sequencing: Efficient targeted sequencing and phasing of the CFTR gene

Background: The sequencing and haplotype phasing of entire gene sequences improves the understanding of the genetic basis of disease and drug response. One example is cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have revolutionized CF treatment, but only in a minority of CF subjects. Observed heterogeneity in CFTR modulator efficacy is related to the range of CFTR mutations; revertant mutations can modify the response to CFTR modulators, and other intronic variations in the ~200 kb CFTR gene have been linked to disease severity. Heterogeneity in the CFTR gene may also be linked to differential responses to CFTR modulators. The Targeted Locus Amplification (TLA) technology from Cergentis can be used to selectively amplify, sequence and phase the entire CFTR gene. With PacBio long-read SMRT Sequencing, TLA amplicons are sequenced intact and long-range phasing information of all fragments in entire amplicons is retrieved. Experimental Design and Methods: The TLA process produces amplicons consisting of 5-10 proximity ligated DNA fragments. TLA was performed on cell line and genomic DNA from Coriell GM12878, which has few heterozygous SNVs in CFTR, and the IB3 cell line, with known haplotypes but heterozygous for the delta508 mutation. All sample types were prepared with high and low density TLA primer sets, targeting coverage of >100 kb of the CFTR gene. Conclusion: We have demonstrated the power and utility of TLA with long-read SMRT Sequencing as a valuable research tool in sequencing and phasing across very long regions of the human genome. This process can be done in an efficient manner, multiplexing multiple genes and samples per SMRT Cell in a process amenable to high-throughput sequencing.


June 1, 2021  |  

Structural variant in the RNA Binding Motif Protein, X-Linked 2 (RBMX2) gene found to be linked to bipolar disorder

Bipolar disorder (BD) is a phenotypically and genetically complex neurological disorder that affects 1% of the worldwide population. There is compelling evidence from family, twin and adoption studies supporting the involvement of a genetic predisposition with estimated heritability up to ~ 80%. The risk in first-degree relatives is ten times higher than in the general population. Linkage and association studies have implicated multiple putative chromosomal loci for BD susceptibility, however no disease genes have yet to be identified. Here, we have fully characterized a ~12 Mb significantly linked (lod score=3.54) genomic region on chromosome Xq24-q27 in an extended family from a genetic isolate that was using long-read single molecule, real-time (SMRT) sequencing. The family segregates BD in at least 4 generations with 16 individuals out of 61 affected. Thus, this family portrays a highly elevated reoccurrence risk compared to the general population. It is expected that the genetic complexity would be reduced in isolated populations, even in genetically complex disorders such as BD, as in the case of this extended family. We selected 16 key individuals from the X-chromosomally linked family to be sequenced. These selected individuals either carried the disease haplotype, were non-carriers of the disease haplotype, or served as married-in controls. We designed a Nimblegen capture array enriching for 5-9 kb fragments spanning the entire 12 Mb region that were then sequenced using long-read SMRT sequencing to screen for causative structural variants (SVs) explaining the increased risk for BD in this extended family. Altogether, 192 SVs were detected in the critically linked region however most of these represented common variants that could be seen across many of the family members regardless of the disease status. One SV stood out that showed perfect segregation among all affected individuals that were carriers of the disease haplotype. This was a 330bp Alu deletion in intron 4 of the RNA Binding Motif Protein, X-Linked 2 (RBMX2) gene that has previously been shown to play a central role in brain development and function. Moreover, Alu elements in general have also previously been associated with at least 37 neurological and neurodegenerative disorders. In order to validate the finding and the functionality of the identified SV further studies like isoform characterization are warranted.


June 1, 2021  |  

Amplification-free protocol for targeted enrichment of repeat expansion genomic regions and SMRT Sequencing

Many genetic disorders are associated with repeat sequence expansions. Obtaining accurate DNA sequence information from these regions will facilitate researchers to further establish the relationship between these genetic disorders and underlying disease mechanisms. Moreover, repeat interruptions have also been shown to act as phenotypic modifiers in some disorders. Targeted sequencing is an economical way to obtain sequence information from one or more defined regions in a genome. However, most targeted enrichment and sequencing methods require some form of DNA amplification. Amplifying large regions with extreme GC content as seen in repeat expansion disorders is challenging and prone to introducing sequence artifacts. DNA amplification also removes any epigenetic signatures present in native DNA. This technique also preserves native DNA molecules for the possibility of direct characterization of epigenetic signatures.


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