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Tuesday, 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…

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Tuesday, 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…

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Tuesday, June 1, 2021

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…

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Tuesday, June 1, 2021

Harnessing kinetic information in Single-Molecule, Real-Time Sequencing.

Single-Molecule Real-Time (SMRT) DNA sequencing is unique in that nucleotide incorporation events are monitored in real time, leading to a wealth of kinetic information in addition to the extraction of the primary DNA sequence. The dynamics of the DNA polymerase that is observed adds an additional dimension of sequence-dependent information, and can be used to learn more about the molecule under study. First, the primary sequence itself can be determined more accurately. The kinetic data can be used to corroborate or overturn consensus calls and even enable calling bases in problematic sequence contexts. Second, using the kinetic information, we can…

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Tuesday, June 1, 2021

Direct sequencing and identification of damaged DNA bases.

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…

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Tuesday, 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…

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Tuesday, June 1, 2021

Single Molecule, Real-Time Sequencing for base modification detection in eukaryotic organisms: Coprinopsis cinerea.

Single Molecule Real-Time (SMRT) DNA sequencing provides a wealth of kinetic information beyond the extraction of the primary DNA sequence, and this kinetic information can provide for the direct detection of modified bases present in genomic DNA. This method has been demonstrated for base modification detection in prokaryotes at base and strand resolutions. In eukaryotes, the common base modifications known to exist are the cytosine variants including methyl, hydroxymethyl, formyl and carboxyl forms. Each of these modifications exhibits different signatures in SMRT kinetic data, allowing for unprecedented possibilities to differentiate between them in direct sequencing data. We present early results…

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Tuesday, 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.…

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Tuesday, 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,…

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Tuesday, 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…

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Tuesday, June 1, 2021

Targeted SMRT Sequencing and phasing using Roche NimbleGen’s SeqCap EZ enrichment

As a cost-effective alternative to whole genome human sequencing, targeted sequencing of specific regions, such as exomes or panels of relevant genes, has become increasingly common. These methods typically include direct PCR amplification of the genomic DNA of interest, or the capture of these targets via probe-based hybridization. Commonly, these approaches are designed to amplify or capture exonic regions and thereby result in amplicons or fragments that are a few hundred base pairs in length, a length that is well-addressed with short-read sequencing technologies. These approaches typically provide very good coverage and can identify SNPs in the targeted region, but…

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Tuesday, June 1, 2021

Full-length HIV-1 env deep sequencing in a donor with broadly neutralizing V1/V2 antibodies.

Background: Understanding the co-evolution of HIV populations and broadly neutralizing antibodies (bNAbs) may inform vaccine design. Novel long-read, next-generation sequencing methods allow, for the first time, full-length deep sequencing of HIV env populations. Methods: We longitudinally examined HIV-1 env populations (12 time points) in a subtype A infected individual from the IAVI primary infection cohort (Protocol C) who developed bNAbs (62% ID50>50 on a diverse panel of 105 viruses) targeting the V1/V2 loop region. We developed a PacBio single molecule, real-time sequencing protocol to deeply sequence full-length env from HIV RNA. Bioinformatics tools were developed to align env sequences, infer…

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Tuesday, June 1, 2021

SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin embedded tissues using adaptive focused acoustics by Covaris.

Recent advances in next-generation sequencing have led to an increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule, Real-Time (SMRT) Sequencing offers a unique advantage for direct analysis of FFPE samples without amplification. However, obtaining ample long-read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. FFPE samples often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and target enrichment. Additionally, the quality and quantity of the recovered DNA vary depending on…

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Tuesday, June 1, 2021

Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human diseases.

The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans. Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete…

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