Alleles of the FMR1 gene with more than 200 CGG repeats generally undergo methylation-coupled gene silencing, resulting in fragile X syndrome, the leading heritable form of cognitive impairment. Smaller expansions (55-200 CGG repeats) result in elevated levels of FMR1 mRNA, which is directly responsible for the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). For mechanistic studies and genetic counseling, it is important to know with precision the number of CGG repeats; however, no existing DNA sequencing method is capable of sequencing through more than ~100 CGG repeats, thus limiting the ability to precisely characterize the disease-causing alleles. The recent development of single molecule, real-time sequencing represents a novel approach to DNA sequencing that couples the intrinsic processivity of DNA polymerase with the ability to read polymerase activity on a single-molecule basis. Further, the accuracy of the method is improved through the use of circular templates, such that each molecule can be read multiple times to produce a circular consensus sequence (CCS). We have succeeded in generating CCS reads representing multiple passes through both strands of repeat tracts exceeding 700 CGGs (>2 kb of 100 percent CG) flanked by native FMR1 sequence, with single-molecule readlengths exceeding 12 kb. This sequencing approach thus enables us to fully characterize the previously intractable CGG-repeat sequence, leading to a better understanding of the distinct associated molecular pathologies. Real-time kinetic data also provides insight into the activity of DNA polymerase inside this unique sequence. The methodology should be widely applicable for studies of the molecular pathogenesis of an increasing number of repeat expansion-associated neurodegenerative and neurodevelopmental disorders, and for the efficient identification of such disorders in the clinical setting.
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.
Sequencing and de novo assembly of the 17q21.31 disease associated region using long reads generated by Pacific Biosciences SMRT Sequencing technology.
Assessment of genome-wide variation revealed regions of the genome with complex, structurally diverse haplotypes that are insufficiently represented in the human reference genome. The 17q21.31 region is one of the most dynamic and complex regions of the human genome. Different haplotypes exist, in direct and inverted orientation, showing evidence of positive selection and predisposing to microdeletion associated with mental retardation. Sequencing of different haplotypes is extremely important to characterize the spectrum of structural variation at this locus. However, de novo assembly with second-generation sequencing reads is still problematic. Using PacBio technology we have sequenced and de novo assembled a tiling path of eight BAC clones (~1.6 Mb region) across this medically relevant region from the library of a hydatidiform mole. Complete hydatidiform moles arise from the fertilization of an enucleated egg from a single sperm and therefore carry a haploid complement of the human genome, eliminating allelic variation that may confound mapping and assembly. The PacBio RS system enables single molecule real time sequencing, featuring long reads and fast turnaround times. With deep sequencing, PacBio reads were able to generate a very uniform sequencing coverage with close to 100% coverage of most of the target interval regions covered. Due to long read lengths, the PacBio RS data could be accurately assembled.
DNA is under constant stress from both endogenous and exogenous sources. DNA base modifications resulting from various types of DNA damage are wide-spread and play important roles in affecting physiological states and disease phenotypes. Examples include oxidative damage (8- oxoguanine, 8-oxoadenine; aging, Alzheimer’s, Parkinson’s), alkylation (1-methyladenine, 6-O- methylguanine; cancer), adduct formation (benzo[a]pyrene diol epoxide (BPDE), pyrimidine dimers; smoking, industrial chemical exposure, chemical UV light exposure, cancer), and ionizing radiation damage (5-hydroxycytosine, 5- hydroxyuracil, 5-hydroxymethyluracil; cancer). Currently, these and other products of DNA damage cannot be sequenced with existing sequencing methods. In contrast, single molecule, real-time (SMRT) DNA sequencing can report on modified DNA bases through an analysis of the DNA polymerase kinetics that is affected by a modified base in the template. We demonstrate the DNA strand-resolved sequencing of over 8 different DNA-damage associated base modifications, with base pair resolution and single DNA molecule sensitivity. We also report on the application of this sequencing capability to biological samples and the development of a generic, open-source algorithm to analyze kinetic information from SMRT sequencing.
Comparative genomics of Shiga toxin-producing Escherichia coli O145:H28 strains associated with the 2007 Belgium and 2010 US outbreaks.
Shiga toxin-producing Escherichia coli (STEC) is an emerging pathogen. Recently there has been a global in the number of outbreaks caused by non-O157 STECs, typically involving six serogroups O26, O45, 0103, 0111, and 0145. STEC O145:H28 has been associated with severe human disease including hemolytic-uremic syndrome (HUS), and is demonstrated by the 2007 Belgian ice-cream-associated outbreak and 2010 US lettuce-associated outbreak, with over 10% of patients developing HUS in each. The goal of this work was to do comparative genomics of strains, clinical and environmental, to investigate genome diversity and virulence evolution of this important foodborne pathogen.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.