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Tuesday, June 1, 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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Tuesday, June 1, 2021

Multiplexing human HLA class I & II genotyping with DNA barcode adapters for high throughput research.

Human MHC class I genes HLA-A, -B, -C, and class II genes HLA-DR, -DP and -DQ, play a critical role in the immune system as major factors responsible for organ transplant rejection. The have a direct or linkage-based association with several diseases, including cancer and autoimmune diseases, and are important targets for clinical and drug sensitivity research. HLA genes are also highly polymorphic and their diversity originates from exonic combinations as well as recombination events. A large number of new alleles are expected to be encountered if these genes are sequenced through the UTRs. Thus allele-level resolution is strongly preferred…

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Tuesday, June 1, 2021

Sequencing complex mixtures of HIV-1 genomes with single-base resolution.

A large number of distinct HIV-1 genomes can be present in a single clinical sample from a patient chronically infected with HIV-1. We examined samples containing complex mixtures of near-full-length HIV-1 genomes. Single molecules were sequenced as near-full-length (9.6 kb) amplicons directly from PCR products without shearing. Mathematical analysis techniques deconvolved the complex mixture of reads into estimates of distinct near-full-length viral genomes with their relative abundances. We correctly estimated the originating genomes to single-base resolution along with their relative abundances for mixtures where the truth was known exactly by independent sequencing methods. Correct estimates were made even when genomes…

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Tuesday, June 1, 2021

Toward comprehensive genomics analysis with de novo assembly.

Whole genome sequencing can provide comprehensive information important for determining the biochemical and genetic nature of all elements inside a genome. The high-quality genome references produced from past genome projects and advances in short-read sequencing technologies have enabled quick and cheap analysis for simple variants. However even with the focus on genome-wide resequencing for SNPs, the heritability of more than 50% of human diseases remains elusive. For non-human organisms, high-contiguity references are deficient, limiting the analysis of genomic features. The long and unbiased reads from single molecule, real-time (SMRT) Sequencing and new de novo assembly approaches have demonstrated the ability…

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Tuesday, June 1, 2021

Whole genome sequencing and epigenome characterization of cancer cells using the PacBio platform.

The comprehensive characterization of cancer genomes and epigenomes for understanding drug resistance remains an important challenge in the field of oncology. For example, PC-9, a non-small cell lung cancer (NSCL) cell line, contains a deletion mutation in exon 19 (DelE746A750) of EGRF that renders it sensitive to erlotinib, an EGFR inhibitor. However, sustained treatment of these cells with erlotinib leads to drug-tolerant cell populations that grow in the presence of erlotinib. However, the resistant cells can be resensitized to erlotinib upon treatment with methyltransferase inhibitors, suggesting a role of epigenetic modification in development of drug resistance. We have characterized for…

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Tuesday, June 1, 2021

Complete resequencing of extended genomic regions using fosmid target capture and single molecule real-time (SMRT) long read sequencing technology.

A longstanding goal of genomic analysis is the identification of causal genetic factors contributing to disease. While the common disease/common variant hypothesis has been tested in many genome-wide association studies, few advancements in identifying causal variation have been realized, and instead recent findings point away from common variants towards aggregate rare variants as causal. A challenge is obtaining complete phased genomic sequences over extended genomic regions from sufficient numbers of cases and controls to identify all potential variation causal of a disease. To address this, we modified methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generaton that…

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Tuesday, June 1, 2021

Un-zipping diploid genomes – revealing all kinds of heterozygous variants from comprehensive hapltotig assemblies

Outside of the simplest cases (haploid, bacteria, or inbreds), genomic information is not carried in a single reference per individual, but rather has higher ploidy (n=>2) for almost all organisms. The existence of two or more highly related sequences within an individual makes it extremely difficult to build high quality, highly contiguous genome assemblies from short DNA fragments. Based on the earlier work on a polyploidy aware assembler, FALCON (https://github.com/PacificBiosciences/FALCON), we developed new algorithms and software (FALCON-unzip) for de novo haplotype reconstructions from SMRT Sequencing data. We apply the algorithms and the prototype software for (1) a highly repetitive diploid…

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Tuesday, June 1, 2021

Profiling the microbiome in fecal microbiota transplantation using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented…

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Tuesday, June 1, 2021

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis

Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. The resulting library can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes using the SMRT Portal assembly program HGAP, plus base modification analysis. The entire process can be done in less than…

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Tuesday, June 1, 2021

Minimization of chimera formation and substitution errors in full-length 16S PCR amplification

The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. One popular, efficient method of profiling communities is to amplify and sequence the evolutionarily conserved 16S rRNA sequence. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost and species-level classification is often not possible. SMRT Sequencing easily spans the entire 1.5 kb 16S gene, and in combination with highly-accurate single-molecule sequences, can improve the identification of individual species in a metapopulation. However, when amplifying…

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Tuesday, June 1, 2021

SMRT Sequencing for the detection of low-frequency somatic variants

The sensitivity, speed, and reduced cost associated with Next-Generation Sequencing (NGS) technologies have made them indispensable for the molecular profiling of cancer samples. For effective use, it is critical that the NGS methods used are not only robust but can also accurately detect low frequency somatic mutations. Single Molecule, Real-Time (SMRT) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays. Here we characterize SMRT…

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Tuesday, June 1, 2021

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue and Zika viruses by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear. Recent publications have highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are…

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Tuesday, June 1, 2021

Immune regions are no longer incomprehensible with SMRT Sequencing

The complex immune regions of the genome, including MHC and KIR, contain large copy number variants (CNVs), a high density of genes, hyper-polymorphic gene alleles, and conserved extended haplotypes (CEH) with enormous linkage disequilibrium (LDs). This level of complexity and inherent biases of short-read sequencing make it challenging for extracting immune region haplotype information from reference-reliant, shotgun sequencing and GWAS methods. As NGS based genome and exome sequencing and SNP arrays have become a routine for population studies, numerous efforts are being made for developing software to extract and or impute the immune gene information from these datasets. Despite these…

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