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June 1, 2021  |  

SMRT Sequencing of whole mitochondrial genomes and its utility in association studies of metabolic disease.

In this study we demonstrate the utility of Single-Molecule Real Time SMRT sequencing to detect variants and to recapitulate whole mitochondrial genomes in an association study of Metabolic syndrome using samples from a well-studied cohort from Micronesia. The Micronesian island of Kosrae is a rare genetic isolate that offers significant advantages for genetic studies of human disease. Kosrae suffers from one of the highest rates of MetS (41%), obesity (52%), and diabetes (17%) globally and has a homogeneous environment making this an excellent population in which to study these significant health problems. We are conducting family-based association analyses aimed at identifying specific mitochondrial variants that contribute to obesity and other co-morbid conditions. We sequenced whole mitochondrial genomes from 10 Kosraen individuals who represent greater than 25 % of the mitochondrial genetic diversity for the entire Kosraen population. Using Pacific Biosciences C2 chemistry, SMRTbell libraries were constructed from pooled, full-length, unsheared 5 kb PCR amplicons, tiling the entire 16.6 kb mtDNA genome. Average read lengths for each sample were between 2500-3000 bp, with 5% of reads between 6,000-8,000 bases, depending on movie lengths. The data generated in this study serve as proof of principle that SMRT Sequencing data can be utilized for identification of high-quality variants and complete mitochondrial genome sequences. These data will be leveraged to identify causative variants for Metabolic syndrome and associated disorders.

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June 1, 2021  |  

Complete HIV-1 genomes from single molecules: Diversity estimates in two linked transmission pairs using clustering and mutual information.

We sequenced complete HIV-1 genomes from single molecules using Single Molecule, Real- Time (SMRT) Sequencing and derive de novo full-length genome sequences. SMRT sequencing yields long-read sequencing results from individual DNA molecules with a rapid time-to-result. These attributes make it a useful tool for continuous monitoring of viral populations. The single-molecule nature of the sequencing method allows us to estimate variant subspecies and relative abundances by counting methods. We detail mathematical techniques used in viral variant subspecies identification including clustering distance metrics and mutual information. Sequencing was performed in order to better understand the relationships between the specific sequences of transmitted viruses in linked transmission pairs. Samples representing HIV transmission pairs were selected from the Zambia Emory HIV Research Project (Lusaka, Zambia) and sequenced. We examine Single Genome Amplification (SGA) prepped samples and samples containing complex mixtures of genomes. Whole genome consensus estimates for each of the samples were made. Genome reads were clustered using a simple distance metric on aligned reads. Appropriate thresholds were chosen to yield distinct clusters of HIV genomes within samples. Mutual information between columns in the genome alignments was used to measure dependence. In silico mixtures of reads from the SGA samples were made to simulate samples containing exactly controlled complex mixtures of genomes and our clustering methods were applied to these complex mixtures. SMRT Sequencing data contained multiple full-length (greater than 9 kb) continuous reads for each sample. Simple whole genome consensus estimates easily identified transmission pairs. The clustering of the genome reads showed diversity differences between the samples, allowing us to characterize the diversity of the individual quasi-species comprising the patient viral populations across the full genome. Mutual information identified possible dependencies of different positions across the full HIV-1 genome. The SGA consensus genomes agreed with prior Sanger sequencing. Our clustering methods correctly segregated reads to their correct originating genome for the synthetic SGA mixtures. The results open up the potential for reference-agnostic and cost effective full genome sequencing of HIV-1.

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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.

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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.

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June 1, 2021  |  

Rapid sequencing of HIV-1 genomes as single molecules from simple and complex samples.

Background: To better understand the relationships among HIV-1 viruses in linked transmission pairs, we sequenced several samples representing HIV transmission pairs from the Zambia Emory HIV Research Project (Lusaka, Zambia) using Single Molecule, Real-Time (SMRT) Sequencing. Methods: Single molecules were sequenced as full-length (9.6 kb) amplicons directly from PCR products without shearing. This resulted in multiple, fully-phased, complete HIV-1 genomes for each patient. We examined Single Genome Amplification (SGA) prepped samples, as well as samples containing complex mixtures of genomes. We detail mathematical techniques used in viral variant subspecies identification, including clustering distance metrics and mutual information, which were used to derive multiple de novo full-length genome sequences for each patient. Whole genome consensus estimates for each sample were made. Genome reads were clustered using a simple distance metric on aligned reads. Appropriate thresholds were chosen to yield distinct clusters of HIV-1 genomes within samples. Mutual information between columns in the genome alignments was used to measure dependence. In silico mixtures of reads from the SGA samples were made to simulate samples containing exactly controlled complex mixtures of genomes and our clustering methods were applied to these complex mixtures. Results: SMRT Sequencing data contained multiple full-length (>9 kb) continuous reads for each sample. Simple whole-genome consensus estimates easily identified transmission pairs. Clustering of genome reads showed diversity differences between samples, allowing characterization of the quasi-species diversity comprising the patient viral populations across the full genome. Mutual information identified possible dependencies of different positions across the full HIV-1 genome. The SGA consensus genomes agreed with prior Sanger sequencing. Our clustering methods correctly segregated reads to their correct originating genome for the synthetic SGA mixtures. Conclusions: SMRT Sequencing yields long-read sequencing results from individual DNA molecules with a rapid time-to-result. These attributes make it a useful tool for continuous monitoring of viral populations. The single-molecule nature of the sequencing method allows us to estimate variant subspecies and relative abundances by counting methods. The results open up the potential for reference-agnostic and cost effective full genome sequencing of HIV-1.

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June 1, 2021  |  

Genomic Architecture of the KIR and MHC-B and -C Regions in Orangutan

PacBio 2013 User Group Meeting Presentation Slides: Lisbeth Guethlein from Stanford University School of Medicine looked at highly repetitive and variable immune regions of the orangutan genome. Guethlein reported that “PacBio managed to accomplish in a week what I have been working on for a couple years” (with Sanger sequencing), and the results were concordant. “Long story short, I was a happy customer.”

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June 1, 2021  |  

Allele-level sequencing and phasing of full-length HLA class I and II genes using SMRT Sequencing technology

The three classes of genes that comprise the MHC gene family are actively involved in determining donor-recipient compatibility for organ transplant, as well as susceptibility to autoimmune diseases via cross-reacting immunization. Specifically, Class I genes HLA-A, -B, -C, and class II genes HLA-DR, -DQ and -DP are considered medically important for genetic analysis to determine histocompatibility. They are highly polymorphic and have thousands of alleles implicated in disease resistance and susceptibility. The importance of full-length HLA gene sequencing for genotyping, detection of null alleles, and phasing is now widely acknowledged. While DNA-sequencing-based HLA genotyping has become routine, only 7% of the HLA genes have been characterized by allele-level sequencing, while 93% are still defined by partial sequences. The gold-standard Sanger sequencing technology is being quickly replaced by second-generation, high- throughput sequencing methods due to its inability to generate unambiguous phased reads from heterozygous alleles. However, although these short, high-throughput, clonal sequencing methods are better at heterozygous allele detection, they are inadequate at generating full-length haploid gene sequences. Thus, full-length gene sequencing from an enhancer-promoter region to a 3’UTR that includes phasing information without the need for imputation still remains a technological challenge. The best way to overcome these challenges is to sequence these genes with a technology that is clonal in nature and has the longest possible read lengths. We have employed Single Molecule Real-Time (SMRT) sequencing technology from Pacific Biosciences for sequencing full-length HLA class I and II genes.

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June 1, 2021  |  

Isoform sequencing: Unveiling the complex landscape of the eukaryotic transcriptome on the PacBio RS II.

Alternative splicing of RNA is an important mechanism that increases protein diversity and is pervasive in the most complex biological functions. While advances in RNA sequencing methods have accelerated our understanding of the transcriptome, isoform discovery remains computationally challenging due to short read lengths. Here, we describe the Isoform Sequencing (Iso-Seq) method using long reads generated by the PacBio RS II. We sequenced rat heart and lung RNA using the Clontech® SMARTer® cDNA preparation kit followed by size selection using agarose gel. Additionally, we tested the BluePippin™ device from Sage Science for efficiently extracting longer transcripts = 3 kb. Post-sequencing, we developed a novel isoform-level clustering algorithm to generate high-quality transcript consensus sequences. We show that our method recovered alternative splice forms as well as alternative stop sites, antisense transcription, and retained introns. To conclude, the Iso-Seq method provides a new opportunity for researchers to study the complex eukaryotic transcriptome even in the absence of reference genomes or annotated transcripts.

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June 1, 2021  |  

Getting the most out of your PacBio libraries with size selection.

PacBio RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and demonstrated consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing. The recently launched P5-C3 chemistry generates even longer reads with N50 often >10,000 bp, making it the best choice for scaffolding and spanning structural rearrangements. With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell library itself. Size selection of a high-quality, sheared 20 kb library using the BluePippin™ System has been demonstrated to increase the N50 read length by as much as 5 kb with C3 chemistry. BluePippin size selection or a more stringent AMPure® PB selection cutoff can be used to recover long fragments from degraded genomic material. The selection of chemistries, P4-C2 versus P5-C3, is highly dependent on the final size distribution of the SMRTbell library and experimental goals. PacBio’s long read lengths also allow for the sequencing of full-length cDNA libraries at single-molecule resolution. However, longer transcripts are difficult to detect due to lower abundance, amplification bias, and preferential loading of smaller SMRTbell constructs. Without size selection, most sequenced transcripts are 1-1.5 kb. Size selection dramatically increases the number of transcripts >1.5 kb, and is essential for >3 kb transcripts.

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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.

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June 1, 2021  |  

Next generation sequencing of full-length HIV-1 env during primary infection.

Background: The use of next generation sequencing (NGS) to examine circulating HIV env variants has been limited due to env’s length (2.6 kb), extensive indel polymorphism, GC deficiency, and long homopolymeric regions. We developed and standardized protocols for isolation, RT-PCR amplification, single molecule real-time (SMRT) sequencing, and haplotype analysis of circulating HIV-1 env variants to evaluate viral diversity in primary infection. Methodology: HIV RNA was extracted from 7 blood plasma samples (1 mL) collected from 5 subjects (one individual sampled and sequenced at 3 time points) in the San Diego Primary Infection Cohort between 3-33 months from their estimated date of infection (EDI). Median viral load per sample was 50,118 HIV RNA copies/mL (range: 22,387-446,683). Full-length (3.2 kb) env amplicons were constructed into SMRTbell templates without shearing, and sequenced on the PacBio RS II using P4/C2 chemistry and 180 minute movie collection without stage start. To examine viral diversity in each sample, we determined haplotypes by clustering circular consensus sequences (CCS), and reconstructing a cluster consensus sequence using a partial order alignment approach. We measured sample diversity both as the mean pairwise distance among reads, and the fraction of reads containing indel polymorphisms. Results: We collected a median of 8,775 CCS reads per SMRT Cell (range: 4243-12234). A median of 7 haplotypes per subject (range: 1-55) were inferred at baseline. For the one subject with longitudinal samples analyzed, we observed an increasing number of distinct haplotypes (8 to 55 haplotypes over the course of 30 months), and an increasing mean pairwise distance among reads (from 0.8% to 1.6%, Tamura-Nei 93). We also observed significant indel polymorphism, with 16% of reads from one sample later in infection (33 months post-EDI) exhibiting deletions of more than 10% of env with respect to the reference strain, HXB2. Conclusions: This study developed a standardized NGS procedure (PacBio SMRT) to deep sequence full-length HIV RNA env variants from the circulating viral population, achieving good coverage, confirming low env diversity during primary infection that increased over time, and revealing significant indel polymorphism that highlights structural variation as important to env evolution. The long, accurate reads greatly simplified downstream bioinformatics analyses, especially haplotype phasing, increasing our confidence in the results. The sequencing methodology and analysis tools developed here could be successfully applied to any area for which full-length HIV env analysis would be useful.

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June 1, 2021  |  

A novel analytical pipeline for de novo haplotype phasing and amplicon analysis using SMRT Sequencing technology.

While the identification of individual SNPs has been readily available for some time, the ability to accurately phase SNPs and structural variation across a haplotype has been a challenge. With individual reads of an average length of 9 kb (P5-C3), and individual reads beyond 30 kb in length, SMRT Sequencing technology allows the identification of mutation combinations such as microdeletions, insertions, and substitutions without any predetermined reference sequence. Long- amplicon analysis is a novel protocol that identifies and reports the abundance of differing clusters of sequencing reads within a single library. Graphs generated via hierarchical clustering of individual sequencing reads are used to generate Markov models representing the consensus sequence of individual clusters found to be significantly different. Long-amplicon analysis is capable of differentiating between underlying sequences that are 99.9% similar, which is suitable for haplotyping and differentiating pseudogenes from coding transcripts. This protocol allows for the identification of structural variation in the MUC5AC gene sequence, despite the presence of a gap in the current genome assembly, and can also be used for HLA haplotyping. Clustering can also been applied to identify full length transcripts for the purpose of estimating consensus sequences and enumerating isoform types. Long-amplicon analysis allows for the elucidation of complex regions otherwise missed by other sequencing technologies, which may contribute to the diagnosis and understanding of otherwise complex diseases.

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June 1, 2021  |  

High-throughput analysis of full-length proviral HIV-1 genomes from PBMCs.

Background: HIV-1 proviruses in peripheral blood mononuclear cells (PBMCs) are felt to be an important reservoir of HIV-1 infection. Given that this pool represents an archival library, it can be used to study virus evolution and CD4+ T cell survival. Accurate study of this pool is burdened by difficulties encountered in sequencing a full-length proviral genome, typically accomplished by assembling overlapping pieces and imputing the full genome. Methodology: Cryopreserved PBMCs collected from a total of 8 HIV+ patients from 1997-2001 were used for genomic DNA extraction. Patients had been receiving cART for 2-8 years at the time samples were obtained. 7 patients had pVL >50 copies/mL (mean: 312,282, range: 18,372-683,400) and 1 had pVL <50. Genomic DNA was subjected to limiting dilution prior to amplification of near-full-length genomes by a newly developed nested PCR. The predicted size of the PCR product was 9.0 kb, spanning from the 5’ LTR through the 3’ LTR. Single molecules were sequenced as near-full-length amplicons directly from PCR products without shearing using commercially available P4-C2 reagents and standard protocols on a PacBio RS II instrument. Quality of the genomes was validated by clonal positive controls and synthetic mixtures. Results: Near-full-length provirus genome sequences were successfully obtained from all 8 patients as continuous long reads from single molecules. PacBio sequencing required approximately 10% of the PCR product needed for Sanger sequencing and generated 325 MB per 3-hour run including 1,800 full-length intact genome reads on average. One patient’s sample was not at a limiting dilution and analysis revealed multiple subspecies. For 8 near-fulllength provirus genomes derived from the other 7 patients, large internal deletions were noted in 2 proviruses; APOBEC-mediated hypermutations were seen in 2 proviruses; and 4 proviruses appeared to be intact genomes. All of the defective proviruses showed a complete absence of resistance mutations in either RT or protease, even after 2-8 years of cART. On the contrary, all of the intact proviruses contained evidence of ART-resistance associated mutations suggesting that they represented relatively recent variants. Conclusions: Combining a novel protocol for full-length limiting dilution amplification of proviruses with PacBio SMRT sequencing allowed for the generation of near-full-length genomes with good quality and an ability to detect minor variants at the 1-10% level. Preliminary data analyses suggest that defective proviruses may represent archival variants that persist long-term in host cells, while intact proviruses within the PBMC pool showing evidence of active virus replication may represent more recent variants.

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June 1, 2021  |  

Long-read, single-molecule applications for protein engineering.

The long read lengths of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases of sequence. This feature is particularly useful in the context of protein engineering, where large numbers of similar constructs are generated routinely to explore the effects of mutations on function and stability. We have developed a PCR-based barcoded sequencing method to generate high quality, full-length sequence data for batches of constructs generated in a common backbone. Individual barcodes are coupled to primers targeting a common region of the vector of interest. The amplified products are pooled into a single DNA library, and sequencing data are clustered by barcode to generate multi-molecule consensus sequences for each construct present in the pool. As a proof-of-concept dataset, we have generated a library of 384 randomly mutated variants of the Phi29 DNA polymerase, a 575 amino acid protein encoded by a 1.7 kb gene. These variants were amplified with a set of barcoded primers, and the resulting library was sequenced on a single SMRT Cell. The data produced sequences that were completely concordant with independent Sanger sequencing, for a 100% accurate reconstruction of the set of clones.

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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.

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