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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.

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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.

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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.

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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.

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June 1, 2021  |  

T-cell receptor profiling using PacBio sequencing of SMARTer libraries

T-cells play a central part in the immune response in humans and related species. T-cell receptors (TCRs), heterodimers located on the T-cell surface, specifically bind foreign antigens displayed on the MHC complex of antigen-presenting cells. The wide spectrum of potential antigens is addressed by the diversity of TCRs created by V(D)J recombination. Profiling this repertoire of TCRs could be useful from, but not limited to, diagnosis, monitoring response to treatments, and examining T-cell development and diversification.

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June 1, 2021  |  

Full-length transcript profiling with the Iso-Seq method for improved genome annotations

Incomplete annotation of genomes represents a major impediment to understanding biological processes, functional differences between species, and evolutionary mechanisms. Often, genes that are large, embedded within duplicated genomic regions, or associated with repeats are difficult to study by short-read expression profiling and assembly. In addition, most genes in eukaryotic organisms produce alternatively spliced isoforms, broadening the diversity of proteins encoded by the genome, which are difficult to resolve with short-read methods. Short-read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. In contrast, Single Molecule, Real-Time (SMRT) Sequencing directly sequences full-length transcripts without the need for assembly and imputation. Here we apply the Iso-Seq method (long-read RNA sequencing) to detect full-length isoforms and the new IsoPhase algorithm to retrieve allele-specific isoform information for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata).

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June 1, 2021  |  

High-throughput SMRT Sequencing of clinically relevant targets

Targeted sequencing with Sanger as well as short read based high throughput sequencing methods is standard practice in clinical genetic testing. However, many applications beyond SNP detection have remained somewhat obstructed due to technological challenges. With the advent of long reads and high consensus accuracy, SMRT Sequencing overcomes many of the technical hurdles faced by Sanger and NGS approaches, opening a broad range of untapped clinical sequencing opportunities. Flexible multiplexing options, highly adaptable sample preparation method and newly improved two well-developed analysis methods that generate highly-accurate sequencing results, make SMRT Sequencing an adept method for clinical grade targeted sequencing. The Circular Consensus Sequencing (CCS) analysis pipeline produces QV 30 data from each single intra-molecular multi-pass polymerase read, making it a reliable solution for detecting minor variant alleles with frequencies as low as 1 %. Long Amplicon Analysis (LAA) makes use of insert spanning full-length subreads originating from multiple individual copies of the target to generate highly accurate and phased consensus sequences (>QV50), offering a unique advantage for imputation free allele segregation and haplotype phasing. Here we present workflows and results for a range of SMRT Sequencing clinical applications. Specifically, we illustrate how the flexible multiplexing options, simple sample preparation methods and new developments in data analysis tools offered by PacBio in support of Sequel System 5.1 can come together in a variety of experimental designs to enable applications as diverse as high throughput HLA typing, mitochondrial DNA sequencing and viral vector integrity profiling of recombinant adeno-associated viral genomes (rAAV).

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June 1, 2021  |  

Comparison of sequencing approaches applied to complex soil metagenomes to resolve proteins of interest

Background: Long-read sequencing presents several potential advantages for providing more complete gene profiling of metagenomic samples. Long reads can capture multiple genes in a single read, and longer reads typically result in assemblies with better contiguity, especially for higher abundance organisms. However, a major challenge with using long reads has been the higher cost per base, which may lead to insufficient coverage of low-abundance species. Additionally, lower single-pass accuracy can make gene discovery for low-abundance organisms difficult. Methods: To evaluate the pros and cons of long reads for metagenomics, we directly compared PacBio and Illumina sequencing on a soil-derived sample, which included spike-in controls of known concentrations of pure referenced samples. For PacBio sequencing, a 10 kb library was sequenced on the Sequel System with 3.0 chemistry. Highly accurate long reads (HiFi reads) with Q20 and higher were generated for downstream analyses using PacBio Circular Consensus Sequencing (CCS) mode. Results were assessed according to the following criteria: DNA extraction capacity, bioinformatics pipeline status, % of proteins with ambiguous AA’s, total unique error-free genes/$1000, total proteins observed in spike-ins/$1000, proteins of interest/$1000, median length of contigs with proteins, and assembly requirements. Results: Both methods had areas of superior performance. DNA extraction capacity was higher for Illumina, the bioinformatics pipeline is well-tested, and there was a lower proportion of proteins with ambiguous AA’s. On the other hand, with PacBio, twice as many unique error-free genes, twice as many total proteins from spike-ins, and ~6 times more proteins of interest were found per $1000 cost. PacBio data produced on average 5 times longer contigs capturing proteins of interest. Additionally, assembly was not required for gene or protein finding, as was the case with Illumina data. Conclusions: In this comparison of PacBio Sequel System with Illumina NextSeq on a complex microbiome, we conclude that the sequencing system of choice may vary, depending on the goals and resources for the project. PacBio sequencing requires a longer DNA extraction method, and the bioinformatics pipeline may require development. On the other hand, the Sequel System generates hundreds of thousands of long HiFi reads per SMRT Cell, producing more genes, more proteins, and longer contigs, thereby offering more information about the metagenomic samples for a lower cost.

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June 1, 2021  |  

Microbiome profiling at the strain level using rRNA amplicons

Strain level microbiome profiling is needed for a full understanding of how microbial communities influence human health. Microbiome profiling of rRNA gene amplicons is a well-understood method that is rapid and inexpensive, but standard 16S rRNA gene methods generally cannot differentiate closely related strains. Whole genome/shotgun microbiome profiling is considered a higher-resolution alternative, but with decreased throughput and significantly increased sequencing costs and analysis burden. With both methods there are also challenges with microbial lysis, DNA preparation, and taxonomic analysis. Specialized microbiome-focused protocols were developed to achieve strain-level taxonomic differentiation using a rapid, high throughput rRNA gene assay. The protocol integrates lysis and DNA preparation improvements with a unique high information content amplicon and associated novel database to enable taxonomic differentiation of closely related microbial strains.

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June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with short-read technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly requires high coverage for error correction, the benefits of unbiased coverage have in the past been lost for low abundance species. SMRT Sequencing performance improvements and the introduction of the Sequel II System has enabled a new, high throughput data type uniquely suited to metagenome characterization: HiFi reads. HiFi reads combine high accuracy with read lengths up to 15 kb, eliminating the need for assembly for most microbiome applications, including functional profiling, gene discovery, and metabolic pathway reconstruction. Here we present the application of the HiFi data type to enable a new method of analyzing metagenomes that does not require assembly.

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June 1, 2021  |  

A complete solution for full-length transcript sequencing using the PacBio Sequel II System

Long read mRNA sequencing methods such as PacBio’s Iso-Seq method offers high-throughput transcriptome profiling in prokaryotic and eukaryotic cells. By avoiding the transcript assembly problem and instead sequencing full-length cDNA, Iso-Seq has emerged as the most reliable technology for annotating isoforms and, in turn, improving proteome predictions in a wide variety of organisms. Improvements in library preparation, sequencing throughput, and bioinformatics has enabled the Iso-Seq method to be complete solution for transcript characterization. The Iso-Seq Express kit is a one-day library prep requiring 60-300 ng of total RNA. The PacBio Sequel II system produces 4-5 million full-length reads, sufficient to profile a whole human transcriptome. Finally, the SQANTI2 software is a powerful tool for categorizing the complex isoforms against reference annotations, while also incorporating orthogonal information such as CAGE peak data, public RNA-seq junction data, and ORF predictions.

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June 1, 2021  |  

New advances in SMRT Sequencing facilitate multiplexing for de novo and structural variant studies

The latest advancements in Sequel II SMRT Sequencing have increased average read lengths up to 50% compared to Sequel II chemistry 1.0 which allows multiplexing of 2-3 small organisms (<500 Mb) such as insects and worms for producing reference quality assemblies, calling structural variants for up to 2 samples with ~3 Gb genomes, analysis of 48 microbial genomes, and up to 8 communities for metagenomic profiling in a single SMRT Cell 8M. With the improved processivity of the new Sequel II sequencing polymerase, more SMRTbell molecules reach rolling circle mode resulting in longer overall read lengths, thus allowing efficient detection of barcodes (up to 80%) in the SMRTbell templates. Multiplexing of genomes larger than microbial organisms is now achievable. In collaboration with the Wellcome Sanger Institute, we have developed a workflow for multiplexing two individual Anopheles coluzzii using as low as 150 ng genomic DNA per individual. The resulting assemblies had high contiguity (contig N50s over 3 Mb) and completeness (>98% of conserved genes) for both individuals. For microbial multiplexing, we multiplexed 48 microbes with varying complexities and sizes ranging 1.6-8.0 Mb in single SMRT Cell 8M. Using a new end-to-end analysis (Microbial Assembly Analysis, SMRT Link 8.0), assemblies resulted in complete circularized genomes (>200-fold coverage) and efficient detection of >3-200 kb plasmids. Finally, the long read lengths (>90 kb) allows detection of barcodes in large insert SMRTbell templates (>15 kb) thus facilitating multiplex of two human samples in 1 SMRT Cell 8M for detecting SVs, Indels and CNVs. Here, we present results and describe workflows for multiplexing samples for specific applications for SMRT Sequencing.

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June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with one sequencing technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly typically requires high coverage for error correction, these benefits have in the past been lost for low-abundance species. The introduction of the Sequel II System has enabled a new, higher throughput, assembly-optional data type that addresses these challenges: HiFi reads. HiFi reads combine QV20 accuracy with long read lengths, eliminating the need for assembly for most metagenome applications, including gene discovery and metabolic pathway reconstruction. In fact, the read lengths and accuracy of HiFi data match or outperform the quality metrics of most metagenome assemblies, enabling cost-effective recovery of intact genes and operons while omitting the resource intensive and data-inefficient assembly step. Here we present the application of HiFi sequencing to both mock and human fecal samples using full-length 16S and shotgun methods. This proof-of-concept work demonstrates the unique strengths of the HiFi method. First, the high correspondence between the expected community composition,16S and shotgun profiling data reflects low context bias. In addition, every HiFi read yields ~5-8 predicted genes, without assembly, using standard tools. If assembly is desired, excellent results can be achieved with Canu and contig binning tools. In summary, HiFi sequencing is a new, cost-effective option for high-resolution functional profiling of metagenomes which complements existing short read workflows.

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June 1, 2021  |  

Low-input single molecule HiFi sequencing for metagenomic samples

HiFi sequencing on the PacBio Sequel II System enables complete microbial community profiling of complex metagenomic samples using whole genome shotgun sequences. With HiFi sequencing, highly accurate long reads overcome the challenges posed by the presence of intergenic and extragenic repeat elements in microbial genomes, thus greatly improving phylogenetic profiling and sequence assembly. Recent improvements in library construction protocols enable HiFi sequencing starting from as low as 5 ng of input DNA. Here, we demonstrate comparative analyses of a control sample of known composition and a human fecal sample from varying amounts of input genomic DNA (1 ug, 200 ng, 5 ng), and present the corresponding library preparation workflows for standard, low input, and Ultra-Low methods. We demonstrate that the metagenome assembly, taxonomic assignment, and gene finding analyses are comparable across all methods for both samples, providing access to HiFi sequencing even for DNA-limited sample types.

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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.

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