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Friday, February 26, 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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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

Rapid full-length Iso-Seq cDNA sequencing of rice mRNA to facilitate annotation and identify splice-site variation.

PacBio’s new Iso-Seq technology allows for rapid generation of full-length cDNA sequences without the need for assembly steps. The technology was tested on leaf mRNA from two model O. sativa ssp. indica cultivars – Minghui 63 and Zhenshan 97. Even though each transcriptome was not exhaustively sequenced, several thousand isoforms described genes over a wide size range, most of which are not present in any currently available FL cDNA collection. In addition, the lack of an assembly requirement provides direct and immediate access to complete mRNA sequences and rapid unraveling of biological novelties.

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Friday, February 26, 2021

Old school/new school genome sequencing: One step backward — a quantum leap forward.

As the costs for genome sequencing have decreased the number of “genome” sequences have increased at a rapid pace. Unfortunately, the quality and completeness of these so–called “genome” sequences have suffered enormously. We prefer to call such genome assemblies as “gene assembly space” (GAS). We believe it is important to distinguish GAS assemblies from reference genome assemblies (RGAs) as all subsequent research that depends on accurate genome assemblies can be highly compromised if the only assembly available is a GAS assembly.

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Friday, February 26, 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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Friday, February 26, 2021

Best practices for whole-genome de novo sequencing with long-read SMRT Sequencing.

With the introduction of P6-C4 chemistry, PacBio has made significant strides with Single Molecule, Real-Time (SMRT) Sequencing . Read lengths averaging between 10 and 15 kb can be now be achieved with extreme reads in the distribution of > 60 kb. The chemistry attains a consensus accuracy of 99.999% (QV50) at 30x coverage which coupled with an increased throughput from the PacBio RS II platform (500 Mb – 1 Gb per SMRT Cell) makes larger genome projects more tractable. These combined advancements in technology deliver results that rival the quality of Sanger “clone-by-clone” sequencing efforts; resulting in closed microbial genomes…

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Friday, February 26, 2021

SMRT Sequencing of the alala genome

Single Molecule Real-Time (SMRT) Sequencing was used to generate long reads for whole genome shotgun sequencing of the genome of the`alala (Hawaiian crow). The ‘alala is endemic to Hawaii, and the only surviving lineage of the crow family, Corvidae, in the Hawaiian Islands. The population declined to less than 20 individuals in the 1990s, and today this charismatic species is extinct in the wild. Currently existing in only two captive breeding facilities, reintroduction of the ‘alala is scheduled to begin in the Fall of 2016. Reintroduction efforts will be assisted by information from the ‘alala genome generated and assembled by…

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Friday, February 26, 2021

Application specific barcoding strategies for SMRT Sequencing

Over the last few years, several advances were implemented in the PacBio RS II System to maximize throughput and efficiency while reducing the cost per sample. The number of useable bases per SMRT Cell now exceeds 1 Gb with the latest P6-C4 chemistry and 6-hour movies. For applications such as microbial sequencing, targeted sequencing, Iso-Seq (full-length isoform sequencing) and Nimblegen’s target enrichment method, current SMRT Cell yields could be an excess relative to project requirements. To this end, barcoding is a viable option for multiplexing samples. For microbial sequencing, multiplexing can be accomplished by tagging sheared genomic DNA during library…

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Friday, February 26, 2021

Multiplexing strategies for microbial whole genome SMRT Sequencing

The increased throughput of the RS II and Sequel Systems enables multiple microbes to be sequenced on a single SMRT Cell. This multiplexing can be readily achieved by simply incorporating a unique barcode for each microbe into the SMRTbell adapters after shearing genomic DNA using a streamlined library construction process. Incorporating a barcode without the requirement for PCR amplification prevents the loss of epigenetic information (e.g., methylation signatures), and the generation of chimeric sequences, while the modified protocol eliminates the need to build several individual SMRTbell libraries. We multiplexed up to 8 unique strains of H. pylori. Each strain was…

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Friday, February 26, 2021

Application-specific barcoding strategies for SMRT Sequencing

The increased sequencing throughput creates a need for multiplexing for several applications. We are here detailing different barcoding strategies for microbial sequencing, targeted sequencing, Iso-Seq full-length isoform sequencing, and Roche NimbleGen’s target enrichment method.

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Friday, February 26, 2021

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome using long-read sequencing

Sequence-based estimation of genetic diversity of Plasmodium falciparum, the most lethal malarial parasite, has proved challenging due to a lack of a complete genomic assembly. The skewed AT-richness (~80.6% (A+T)) of its genome and the lack of technology to assemble highly polymorphic sub-telomeric regions that contain clonally variant, multigene virulence families (i.e. var and rifin) have confounded attempts using short-read NGS technologies. Using single molecule, real-time (SMRT) sequencing, we successfully compiled all 14 nuclear chromosomes of the P. falciparum genome from telomere-to-telomere in single contigs. Specifically, amplification-free sequencing generated reads of average length 12 kb, with =50% of the reads…

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Friday, February 26, 2021

Characterizing haplotype diversity at the immunoglobulin heavy chain locus across human populations using novel long-read sequencing and assembly approaches

The human immunoglobulin heavy chain locus (IGH) remains among the most understudied regions of the human genome. Recent efforts have shown that haplotype diversity within IGH is elevated and exhibits population specific patterns; for example, our re-sequencing of the locus from only a single chromosome uncovered >100 Kb of novel sequence, including descriptions of six novel alleles, and four previously unmapped genes. Historically, this complex locus architecture has hindered the characterization of IGH germline single nucleotide, copy number, and structural variants (SNVs; CNVs; SVs), and as a result, there remains little known about the role of IGH polymorphisms in inter-individual…

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Friday, February 26, 2021

T-cell receptor profiling using PacBio sequencing of SMARTer libraries

T-cells play a central part in the immune response in humans and related species. T-cell receptors (TCRs), heterodimers located on the T-cell surface, specifically bind foreign antigens displayed on the MHC complex of antigen-presenting cells. The wide spectrum of potential antigens is addressed by the diversity of TCRs created by V(D)J recombination. Profiling this repertoire of TCRs could be useful from, but not limited to, diagnosis, monitoring response to treatments, and examining T-cell development and diversification.

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Friday, February 26, 2021

Mitochondrial DNA sequencing using PacBio SMRT technology

Mitochondrial DNA (mtDNA) is a compact, double-stranded circular genome of 16,569 bp with a cytosine-rich light (L) chain and a guanine-rich heavy (H) chain. mtDNA mutations have been increasingly recognized as important contributors to an array of human diseases such as Parkinson’s disease, Alzheimer’s disease, colorectal cancer and Kearns–Sayre syndrome. mtDNA mutations can affect all of the 1000-10,000 copies of the mitochondrial genome present in a cell (homoplasmic mutation) or only a subset of copies (heteroplasmic mutation). The ratio of normal to mutant mtDNAs within cells is a significant factor in whether mutations will result in disease, as well as…

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