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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.


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, high-accuracy reads (SMRT (circular consensus sequencing) CCS or second-generation reads) to generate long, highly accurate reads that are then used for assembly. We have developed a new paradigm for microbial de novo assemblies in which long SMRT sequencing reads (average readlengths >5,000 bases) 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%) to 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. The process has been automated and requires less than 1 day from an unknown DNA sample to its complete de novo genome and epigenome.


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.


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.”


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  |  

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.


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.


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.


June 1, 2021  |  

Using whole exome sequencing and bacterial pathogen sequencing to investigate the genetic basis of pulmonary non-tuberculous mycobacterial infections.

Pulmonary non-tuberculous mycobacterial (PNTM) infections occur in patients with chronic lung disease, but also in a distinct group of elderly women without lung defects who share a common body morphology: tall and lean with scoliosis, pectus excavatum, and mitral valve prolapse. In order to characterize the human host susceptibility to PNTM, we performed whole exome sequencing (WES) of 44 individuals in extended families of patients with active PNTM as well as 55 additional unrelated individuals with PNTM. This unique collection of familial cohorts in PNTM represents an important opportunity for a high yield search for genes that regulate mucosal immunity. An average of 58 million 100bp paired-end Illumina reads per exome were generated and mapped to the hg19 reference genome. Following variant detection and classification, we identified 58,422 potentially high-impact SNPs, 97.3% of which were missense mutations. Segregating variants using the family pedigrees as well as comparisons to the unrelated individuals identified multiple potential variants associated with PNTM. Validations of these candidate variants in a larger PNTM cohort are underway. In addition to WES, we sequenced the genomes of 52 mycobacterial isolates, including 9 from these PNTM patients, to integrate host PNTM susceptibility with mycobacterial genotypes and gain insights into the key factors involved in this devastating disease. These genomes were sequenced using a combination of 454, Illumina, and PacBio platforms and assembled using multiple genome assemblers. The resulting genome sequences were used to identify mycobacterial genotypes associated with virulence, invasion, and drug resistance.


June 1, 2021  |  

New discoveries from closing Salmonella genomes using Pacific Biosciences continuous long reads.

The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition, we were able to detect novel methyltransferases (MTases) by using the Pacific Biosciences kinetic score distributions showing that each serovar appears to have a novel methylation pattern. For example while all Salmonella serovars examined so far have methylase specific activity for 5’-GATC-3’/3’-CTAG-5’ and 5’-CAGAG-3’/3’-GTCTC-5’ (underlined base indicates a modification), S. Heidelberg is uniquely specific for 5’-ACCANCC-3’/3’-TGGTNGG-5’, while S. Typhimurium has uniquely methylase specific for 5′-GATCAG-3’/3′- CTAGTC-5′ sites, for the samples examined so far. We believe that this may be due to the unique environments and phages that these serotypes have been exposed to. Furthermore, our analysis identified and closed a variety of plasmids such as mobilization plasmids, antimicrobial resistance plasmids and IncX plasmids carrying a Type IV secretion system (T4SS). The VirB/D4 T4SS apparatus is important in that it assists with rapid dissemination of antibiotic resistance and virulence determinants. Presently, only limited information exists regarding the genotypic characterization of drug resistance in S. Heidelberg isolates derived from various host species. Here, we characterize two S. Heidelberg outbreak isolates from two different outbreaks. Both isolates contain the IncX plasmid of approximately 35 kb, and carried the genes virB1, virB2, virB3/4, virB5, virB6, virB7, virB8, virB9, virB10, virB11, virD2, and virD4, that are associated with the T4SS. In addition, the outbreak isolate associated with ground turkey carries a 4,473 bp mobilization plasmid and an incompatibility group (Inc) I1 antimicrobial resistance plasmid encoding resistance to gentamicin (aacC2), beta-lactam (bl2b_tem), streptomycin (aadAI) and tetracycline (tetA, tetR) while the outbreak isolate associated with chicken breast carries the IncI1 plasmid encoding resistance to gentamicin (aacC2), streptomycin (aadAI) and sulfisoxazole (sul1). Using this new technology we explored the genetic elements present in resistant pathogens which will achieve a better understanding of the evolution of Salmonella.


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.


June 1, 2021  |  

Characterization of NNRTI mutations in HIV-1 RT using Single Molecule, Real-Time SMRT Sequencing.

Background: Genotypic testing of chronic viral infections is an important part of patient therapy and requires assays capable of detecting the entire spectrum of viral mutations. Single Molecule, Real-Time (SMRT) sequencing offers several advantages to other sequencing technologies, including superior resolution of mixed populations and long read lengths capable of spanning entire viral protein coding regions. We examined detection sensitivity of SMRT sequencing using a mixture of HIV-1 RT gene coding regions containing single NNRTI mutations. Methodology: SMRTbell templates were prepared from PCR products generated from a prospective reference material being developed by BC Center of Excellence for HIV/AIDS, and contained a mixture of fifteen infectious viruses containing single NNRTI resistance mutations (viz V90I, K101E, K103N, V108I, E138A/G/K/Q, V179D, Y181C, Y188C, G190A/S, M230L and P236L) built upon the HIV-1LAI molecular clone. Templates were sequenced on the PacBio RS II to obtain single molecule long reads using P4/C2 chemistry, using 180 minute movie collection without stage start. The relative abundances of the mutant viruses were then estimated using codon-aware analysis methods. Results: Sequencing of these templates produced average read lengths of 5.0 KB, comprising 40,000-fold coverage across the entire amplicon per SMRT Cell. All the expected mutations in the mixture of mutant viruses were accurately identified. Frequencies of NNRTI variants estimated ranged from 0.5% to 12.5%. Conclusions: Codon analysis revealed a number of variants across the amplicon with highly consistent results across SMRT Cells. From a single SMRT Cell, variants were accurately and reliably detected down to 0.5% with simple analyses. Long polymerase reads and high accuracy reads make it possible to call variants from just a few molecules. SMRT Sequencing can identify species comprising a mixed viral population, with granularity and low cost of consumables allowing for smaller multiplexing of samples and first-in-first-out processing.


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.


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.


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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