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June 1, 2021  |  

Evaluating the potential of new sequencing technologies for genotyping and variation discovery in human data.

A first look at Pacific Biosciences RS data Pacific Biosciences technology provides a fundamentally new data type that provides the potential to overcome these limitations by providing significantly longer reads (now averaging >1kb), enabling more unique seeds for reference alignment. In addition, the lack of amplification in the library construction step avoids a common source of base composition bias. With these potential advantages in mind, we here evaluate the utility of the Pacific Biosciences RS platform for human medical resequencing projects by assessing the quality of the raw sequencing data, as well as its use for SNP discovery and genotyping using the Genome Analysis Toolkit (GATK).

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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  |  

Single Molecule Real-Time (SMRT) Sequencing of genes implicated in autosomal recessive diseases.

In today’s clinical diagnostic laboratories, the detection of the disease causing mutations is either done through genotyping or Sanger sequencing. Whether done singly or in a multiplex assay, genotyping works only if the exact molecular change is known. Sanger sequencing is the gold standard method that captures both known and novel molecular changes in the disease gene of interest. Most clinical Sanger sequencing assays involve PCR-amplifying the coding sequences of the disease target gene followed by bi-directional sequencing of the amplified products. Therefore for every patient sample, one generates multiple amplicons singly and each amplicon leads to two separate sequencing reactions. Single Molecule, Real-Time (SMRT) sequencing offers several advantages to Sanger sequencing including long read lengths, first-in-first-out processing, fast time to result, high-levels of multiplexing and substantially reduced costs. For our first proof-of-concept experiment, we queried 3 known disease-associated mutations in de-identified clinical samples. We started off with 3 autosomal recessive diseases found at an increased frequency in the Ashkenazi Jewish population: Tay Sachs disease, Niemann-Pick disease and Canavan disease. The mutated gene in Tays Sachs is HEXA, Niemann-Pick is SMPD1 and Canavan is ASPA. Coding exons were amplified in multiple (6-13) amplicons for each gene from both non-carrier and carriers. Amplicons were purified, concentrations normalized, and combined prior to SMRTbell™ Library prep. A single SMRTbell library was sequenced for each gene from each patient using standard Pacific Biosciences C2 chemistry and protocols. Average read lengths of 4,000 bp across samples allowed for high-quality Circular Consensus Sequences (CCS) across all amplicons (all less than 1 kb). This high quality CCS data permitted the clean partitioning of reads from a patient in the presence of heterozygous events. Using non-carrier sequencing as a control, we were able to correctly identify the known events in carrier genes. This suggests the potential utility of SMRT sequencing in a clinical setting, enabling a cost-effective method of replacing targeted mutation detection with sequencing of the entire gene.

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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  |  

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  |  

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.

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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  |  

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.

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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.

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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.

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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  |  

SMRT Sequencing solutions for investigative studies to understand evolutionary processes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers to understand molecular mechanisms in evolution and gain insight into adaptive strategies. With read lengths exceeding 10 kb, we are able to sequence high-quality, closed microbial genomes with associated plasmids, and investigate large genome complexities, such as long, highly repetitive, low-complexity regions and multiple tandem-duplication events. Improved genome quality, observed at 99.9999% (QV60) consensus accuracy, and significant reduction of gap regions in reference genomes (up to and beyond 50%) allow researchers to better understand coding sequences with high confidence, investigate potential regulatory mechanisms in noncoding regions, and make inferences about evolutionary strategies that are otherwise missed by the coverage biases associated with short- read sequencing technologies. Additional benefits afforded by SMRT Sequencing include the simultaneous capability to detect epigenomic modifications and obtain full-length cDNA transcripts that obsolete the need for assembly. With direct sequencing of DNA in real-time, this has resulted in the identification of numerous base modifications and motifs, which genome-wide profiles have linked to specific methyltransferase activities. Our new offering, the Iso-Seq Application, allows for the accurate differentiation between transcript isoforms that are difficult to resolve with short-read technologies. PacBio reads easily span transcripts such that both 5’/3’ primers for cDNA library generation and the poly-A tail are observed. As such, exon configuration and intron retention events can be analyzed without ambiguity. This technological advance is useful for characterizing transcript diversity and improving gene structure annotations in reference genomes. We review solutions available with SMRT Sequencing, from targeted sequencing efforts to obtaining reference genomes (>100 Mb). This includes strategies for identifying microsatellites and conducting phylogenetic comparisons with targeted gene families. We highlight how to best leverage our long reads that have exceeded 20 kb in length for research investigations, as well as currently available bioinformatics strategies for analysis. Benefits for these applications are further realized with consistent use of size selection of input sample using the BluePippin™ device from Sage Science as demonstrated in our genome improvement projects. Using the latest P5-C3 chemistry on model organisms, these efforts have yielded an observed contig N50 of ~6 Mb, with the longest contig exceeding 12.5 Mb and an average base quality of QV50.

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June 1, 2021  |  

Genomic DNA sequences of HLA class I alleles generated using multiplexed barcodes and SMRT DNA Sequencing technology.

Allelic-level resolution HLA typing is known to improve survival prognoses post Unrelated Donor (UD) Haematopoietic Stem Cell Transplantation (HSCT). Currently, many commonly used HLA typing methodologies are limited either due to the fact that ambiguity cannot be resolved or that they are not amenable to high-throughput laboratories. Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology enables sequencing of single molecules in isolation and has read-length capabilities to enable whole gene sequencing for HLA. DNA barcode technology labels samples with unique identifiers that can be traced throughout the sequencing process. The use of DNA barcodes means that multiple samples can be sequenced in a single experiment but data can still be attributed to the correct sample. Here we describe the results of experiments that use DNA barcodes to facilitate sequencing of multiple samples for full-length HLA class I genes (known as multiplexing).

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June 1, 2021  |  

HLA sequencing using SMRT Technology – High resolution and high throughput HLA genotyping in a clinical setting

Sequence based typing (SBT) is considered the gold standard method for HLA typing. Current SBT methods are rather laborious and are prone to phase ambiguity problems and genotyping uncertainties. As a result, the NGS community is rapidly seeking to remedy these challenges, to produce high resolution and high throughput HLA sequencing conducive to a clinical setting. Today, second generation NGS technologies are limited in their ability to yield full length HLA sequences required for adequate phasing and identification of novel alleles. Here we present the use of single molecule real time (SMRT) sequencing as a means of determining full length/long HLA sequences. Moreover we reveal the scalability of this method through multiplexing approches and determine HLA genotyping calls through the use of third party Gendx NGSengine® software.

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