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Friday, February 26, 2021

Sequencing of expanded CGG repeats in the FMR1 gene.

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…

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Friday, February 26, 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…

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Friday, February 26, 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…

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Friday, February 26, 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…

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Friday, February 26, 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…

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Friday, February 26, 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…

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Friday, February 26, 2021

SMRT Sequencing solutions for large genomes and transcriptomes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers in large genome complexities, such as long, highly repetitive, low-complexity regions and duplication events, and differentiating between transcript isoforms that are difficult to resolve with short-read technologies. We present solutions available for both reference genome improvement (>100 MB) and transcriptome research to best leverage long reads that have exceeded 20 Kb in length. Benefits for these applications are further realized with consistent use of size-selection of input sample using the BluePippin™ device from Sage Science. Highlights from our genome assembly projects using the latest P5-C3 chemistry on model organisms…

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Friday, February 26, 2021

A comparison of assemblers and strategies for complex, large-genome sequencing with PacBio long reads.

PacBio sequencing holds promise for addressing large-genome complexities, such as long, highly repetitive, low-complexity regions and duplication events that are difficult to resolve with short-read technologies. Several strategies, with varying outcomes, are available for de novo sequencing and assembling of larger genomes. Using a diploid fungal genome, estimated to be ~80 Mb in size, as the basis dataset for comparison, we highlight assembly options when using only PacBio sequencing or a combined strategy leveraging data sets from multiple sequencing technologies. Data generated from SMRT Sequencing was subjected to assembly using different large-genome assemblers, and comparisons of the results will be…

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Friday, February 26, 2021

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

The long read lengths of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases of sequence. This feature is particularly useful in the context of protein engineering, where large numbers of similar constructs are generated routinely to explore the effects of mutations on function and stability. We have developed a PCR-based barcoded sequencing method to generate high quality, full-length sequence data for batches of constructs generated in a common backbone. Individual barcodes are coupled to primers targeting a common region of the vector of interest. The amplified products are pooled into a single DNA library, and sequencing data…

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Friday, February 26, 2021

Accurately surveying uncultured microbial species with SMRT Sequencing

Background: Microbial ecology is reshaping our understanding of the natural world by revealing the large phylogenetic and functional diversity of microbial life. However the vast majority of these microorganisms remain poorly understood, as most cultivated representatives belong to just four phylogenetic groups and more than half of all identified phyla remain uncultivated. Characterization of this microbial ‘dark matter’ will thus greatly benefit from new metagenomic methods for in situ analysis. For example, sensitive high throughput methods for the characterization of community composition and structure from the sequencing of conserved marker genes. Methods: Here we utilize Single Molecule Real-Time (SMRT) sequencing…

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Friday, February 26, 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…

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Friday, February 26, 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…

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Friday, February 26, 2021

SMRT Sequencing solutions for plant genomes and transcriptomes

Single Molecule, Real-Time (SMRT) Sequencing provides efficient, streamlined solutions to address new frontiers in plant genomes and transcriptomes. Inherent challenges presented by highly repetitive, low-complexity regions and duplication events are directly addressed with multi- kilobase read lengths exceeding 8.5 kb on average, with many exceeding 20 kb. Differentiating between transcript isoforms that are difficult to resolve with short-read technologies is also now possible. We present solutions available for both reference genome and transcriptome research that best leverage long reads in several plant projects including algae, Arabidopsis, rice, and spinach using only the PacBio platform. Benefits for these applications are further…

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Friday, February 26, 2021

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing.

Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective procedure to detect adenomas, the precursors to CRC, is colonoscopy, which reduces CRC incidence by 80%. However, it is an invasive approach that is unpleasant for the patient, expensive, and poses some risk of complications such as colon perforation. A non-invasive screening approach with detection rates comparable to those of colonoscopy has not yet been established. The current study applies Pacific Biosciences third generation, single molecule sequencing to the inspection…

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Friday, February 26, 2021

Evaluation of multiplexing strategies for HLA genotyping using PacBio Sequencing technology.

Fully phased allele-level sequencing of highly polymorphic HLA genes is greatly facilitated by SMRT Sequencing technology. In the present work, we have evaluated multiple DNA barcoding strategies for multiplexing several loci from multiple individuals, using three different tagging methods. Specifically MHC class I genes HLA-A, -B, and –C were indexed via DNA Barcodes by either tailed primers or barcoded SMRTbell adapters. Eight different 16-bp barcode sequences were used in symmetric & asymmetric pairing. Eight DNA barcoded adapters in symmetric pairing were independently ligated to a pool of HLA-A, -B and –C for eight different individuals, one at a time and…

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