Menu
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  |  

Collection of major HLA allele sequences in Japanese population toward the precise NGS based HLA DNA typing at the field 4 level

We previously reported on the use of the Ion PGM next generation sequencing (NGS) platform to genotype HLA class I and class II genes by a super-high resolution, single-molecule, sequence-based typing (SS-SBT) method (Shiina et al. 2012). However, HLA alleles could not be assigned at the field 4 level at some HLA loci such as DQA1, DPA1 and DPB1 because the SNP and indel densities were too low to identify and separate both of the phases. In this regard, we have now added the single molecule, real-time (SMRT) DNA sequencer PacBio RS II method to our analysis in order to test whether it might determine the HLA allele sequences in some of the loci with which we previously had difficulties. In this study, we report on sequence-based genotyping of entire HLA gene sequences from the promoter-enhancer region to 3’UTR of the major HLA loci (A, B, C, DRB1, DRB345, DQA1, DQB1, DPA1 and DPB1) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the HLA loci and the PacBio RS II and Ion PGM systems.


June 1, 2021  |  

Characterizing haplotype diversity at the immunoglobulin heavy chain locus across human populations using novel long-read sequencing and assembly approaches

The human immunoglobulin heavy chain locus (IGH) remains among the most understudied regions of the human genome. Recent efforts have shown that haplotype diversity within IGH is elevated and exhibits population specific patterns; for example, our re-sequencing of the locus from only a single chromosome uncovered >100 Kb of novel sequence, including descriptions of six novel alleles, and four previously unmapped genes. Historically, this complex locus architecture has hindered the characterization of IGH germline single nucleotide, copy number, and structural variants (SNVs; CNVs; SVs), and as a result, there remains little known about the role of IGH polymorphisms in inter-individual antibody repertoire variability and disease. To remedy this, we are taking a multi-faceted approach to improving existing genomic resources in the human IGH region. First, from whole-genome and fosmid-based datasets, we are building the largest and most ethnically diverse set of IGH reference assemblies to date, by employing PacBio long-read sequencing combined with novel algorithms for phased haplotype assembly. In total, our effort will result in the characterization of >15 phased haplotypes from individuals of Asian, African, and European descent, to be used as a representative reference set by the genomics and immunogenetics community. Second, we are utilizing this more comprehensive sequence catalogue to inform the design and analysis of novel targeted IGH genotyping assays. Standard targeted DNA enrichment methods (e.g., exome capture) are currently optimized for the capture of only very short (100’s of bp) DNA segments. Our platform uses a modified bench protocol to pair existing capture-array technologies with the enrichment of longer fragments of DNA, enabling the use of PacBio sequencing of DNA segments up to 7 Kb. This substantial increase in contiguity disambiguates many of the complex repeated structures inherent to the locus, while yielding the base pair fidelity required to call SNVs. Together these resources will establish a stronger framework for further characterizing IGH genetic diversity and facilitate IGH genomic profiling in the clinical and research settings, which will be key to fully understanding the role of IGH germline variation in antibody repertoire development and disease.


June 1, 2021  |  

Profiling complex population genomes with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS sequencing enables functional profiling as well, with the ultimate goal of complete genome assemblies. Here we compare the complex microbiomes in 5 cow rumen samples, for which Illumina WGS sequence data was also available. To maximize the PacBio single-molecule sequence accuracy, libraries of 2 to 3 kb were generated, allowing many polymerase passes per molecule. The resulting reads were filtered at predicted single-molecule accuracy levels up to 99.99%. Community compositions of the 5 samples were compared with Illumina WGS assemblies from the same set of samples, indicating rare organisms were often missed with Illumina. Assembly from PacBio CCS reads yielded a contig >100 kb in length with 6-fold coverage. Mapping of Illumina reads to the 101 kb contig verified the PacBio assembly and contig sequence. These results illustrate ways in which long accurate reads benefit analysis of complex communities.


June 1, 2021  |  

Profiling complex communities with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS sequencing enables functional profiling as well, with the ultimate goal of complete genome assemblies. Here we compare the complex microbiomes in 5 cow rumen samples, for which Illumina WGS sequence data was also available. To maximize the PacBio single-molecule sequence accuracy, libraries of 2 to 3 kb were generated, allowing many polymerase passes per molecule. The resulting reads were filtered at predicted single-molecule accuracy levels up to 99.99%. Community compositions of the 5 samples were compared with Illumina WGS assemblies from the same set of samples, indicating rare organisms were often missed with Illumina. Assembly from PacBio CCS reads yielded a contig >100 kb in length with 6-fold coverage. Mapping of Illumina reads to the 101 kb contig verified the PacBio assembly and contig sequence. Scaffolding with reads from a PacBio unsheared library produced a complete genome of 2.4 Mb. These results illustrate ways in which long accurate reads benefit analysis of complex communities.


June 1, 2021  |  

Using the PacBio Sequel System to taxonomically and functionally classify metagenomic samples in a trial of patients undergoing fecal microbiota transplantation

Whole-sample shotgun sequencing can provide a more detailed view of a metagenomic community than 16S sequencing, but its use in multi-sample experiments is limited by throughput, cost and analysis complexity. While short-read sequencing technologies offer higher throughput, read lengthss less fewer than 500 bp will rarely cover a gene of interest, and necessitate assembly before further analysis. Assembling large fragments requires sampling each community member at a high depth, significantly increasing the amount of sequencing needed, and limiting the analysis of rare community members. Assembly methods also risk It is also possible to incorrectly combine combining sequences from different community members.


June 1, 2021  |  

SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants

Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being developed as a predictive biomarker of primary resistance to androgen receptor (AR)-targeted therapies in CRPC. Multiple additional AR-V species are expressed in clinical CRPC, but the extent to which these may be co-expressed with AR-V7 or predict resistance is not known.


June 1, 2021  |  

Characterizing the pan-genome of maize with PacBio SMRT Sequencing

Maize is an amazingly diverse crop. A study in 20051 demonstrated that half of the genome sequence and one-third of the gene content between two inbred lines of maize were not shared. This diversity, which is more than two orders of magnitude larger than the diversity found between humans and chimpanzees, highlights the inability of a single reference genome to represent the full pan-genome of maize and all its variants. Here we present and review several efforts to characterize the complete diversity within maize using the highly accurate long reads of PacBio Single Molecule, Real-Time (SMRT) Sequencing. These methods provide a framework for a pan-genomic approach that can be applied to studies of a wide variety of important crop species.


June 1, 2021  |  

A complete solution for high-quality genome annotation using the PacBio Iso-Seq method

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more transcripts. Most importantly, the method is supported by a maturing suite of official and community-developed tools. The SMRT Link Iso-Seq application outputs high-quality (>99% accurate), full-length transcript sequences that can optionally be mapped to a reference genome for a single SMRT Cell worth of data in 6-9 hours. For example, the SQANTI2 tool classifies Iso-Seq transcripts against a reference annotation, filters potential library artifacts, and processes information from both long read-only and short read-based quantification. IsoPhase is a tool for identifying allele-specific isoform expression. Cogent has been used to process Iso-Seq transcripts in a genome-independent manner to assess genome assemblies. Finally, IsoAnnot is an up-and-coming tool for identifying differential isoform expression across different samples. We describe how these tools complement each other and provide guidelines to make the best use out of Iso-Seq data for understanding transcriptomes.


June 1, 2021  |  

Metagenomic analysis of type II diabetes gut microbiota using PacBio HiFi reads reveals taxonomic and functional differences

In the past decade, the human microbiome has been increasingly shown to play a major role in health. For example, imbalances in gut microbiota appear to be associated with Type II diabetes mellitus (T2DM) and cardiovascular disease. Coronary artery disease (CAD) is a major determinant of the long-term prognosis among T2DM patients, with a 2- to 4-fold increased mortality risk when present. However, the exact microbial strains or functions implicated in disease need further investigation. From a large study with 523 participants (185 healthy controls, 186 T2DM patients without CAD, and 106 T2DM patients with CAD), 3 samples from each patient group were selected for long read sequencing. Each sample was prepared and sequenced on one Sequel II System SMRT Cell, to assess whether long accurate PacBio HiFi reads could yield additional insights to those made using short reads. Each of the 9 samples was subject to metagenomic assembly and binning, taxonomic classification and functional profiling. Results from metagenomic assembly and binning show that it is possible to generate a significant number of complete MAGs (Metagenome Assembled Genomes) from each sample, with over half of the high-quality MAGs being represented by a single circular contig. We show that differences found in taxonomic and functional profiles of healthy versus diabetic patients in the small 9-sample study align with the results of the larger study, as well as with results reported in literature. For example, the abundances of beneficial short- chain fatty acid (SCFA) producers such as Phascolarctobacterium faecium and Faecalibacterium prausnitzii were decreased in T2DM gut microbiota in both studies, while the abundances of quinol and quinone biosynthesis pathways were increased as compared to healthy controls. In conclusion, metagenomic analysis of long accurate HiFi reads revealed important taxonomic and functional differences in T2DM versus healthy gut microbiota. Furthermore, metagenome assembly of long HiFi reads led to the recovery of many complete MAGs and a significant number of complete circular bacterial chromosome sequences.


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.