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Wednesday, June 23, 2021

Application Brief: Targeted sequencing for amplicons – Best Practices

With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel System, you can easily and cost effectively generate highly accurate long reads (HiFi reads, >99% single-molecule accuracy) from genes or regions of interest ranging in size from several hundred base pairs to 20 kb. Target all types of variation across relevant genomic regions, including low complexity regions like repeat expansions, promoters, and flanking regions of transposable elements.

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Tuesday, June 1, 2021

Unique haplotype structure determination in human genome using Single Molecule, Real-Time (SMRT) Sequencing of targeted full-length fosmids.

Determination of unique individual haplotypes is an essential first step toward understanding how identical genotypes having different phases lead to different biological interpretations of function, phenotype, and disease. Genome-wide methods for identifying individual genetic variation have been limited in their ability to acquire phased, extended, and complete genomic sequences that are long enough to assemble haplotypes with high confidence. We explore a recombineering approach for isolation and sequencing of a tiling of targeted fosmids to capture interesting regions from human genome. Each individual fosmid contains large genomic fragments (~35?kb) that are sequenced with long-read SMRT technology to generate contiguous long…

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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

Highly sensitive and cost-effective detection of BRCA1 and BRCA2 cancer variants in FFPE samples using Multiplicom’s MASTR technology & Single Molecule, Real-Time (SMRT) Sequencing

Specific mutations in BRCA1 and BRCA2 have been shown to be associated with several types of cancers. Molecular profiling of cancer samples requires assays capable of accurately detecting the entire spectrum of variants, including those at relatively low frequency. Next-Generation Sequencing (NGS) has been a powerful tool for researchers to better understand cancer genetics. Here we describe a targeted re-sequencing workflow that combines barcoded amplification of BRCA1 and BRCA2 exons from 12 FFPE tumor samples using Multiplicom’s MASTR technology with PacBio SMRT Sequencing. This combination allows for the accurate detection of variants in a cost-effective and timely manner.

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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

Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing

Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust, but capable of accurately detecting 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 for studying cancer variants. Here we…

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Tuesday, June 1, 2021

Resolving KIR genotypes and haplotypes simultaneously using Single Molecule, Real-Time Sequencing

The killer immunoglobulin-like receptors (KIR) genes belong to the immunoglobulin superfamily and are widely studied due to the critical role they play in coordinating the innate immune response to infection and disease. Highly accurate, contiguous, long reads, like those generated by SMRT Sequencing, when combined with target-enrichment protocols, provide a straightforward strategy for generating complete de novo assembled KIR haplotypes. We have explored two different methods to capture the KIR region; one applying the use of fosmid clones and one using Nimblegen capture.

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Tuesday, June 1, 2021

Multiplex target enrichment using barcoded multi-kilobase fragments and probe-based capture technologies

Target enrichment capture methods allow scientists to rapidly interrogate important genomic regions of interest for variant discovery, including SNPs, gene isoforms, and structural variation. Custom targeted sequencing panels are important for characterizing heterogeneous, complex diseases and uncovering the genetic basis of inherited traits with more uniform coverage when compared to PCR-based strategies. With the increasing availability of high-quality reference genomes, customized gene panels are readily designed with high specificity to capture genomic regions of interest, thus enabling scientists to expand their research scope from a single individual to larger cohort studies or population-wide investigations. Coupled with PacBio® long-read sequencing, these…

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Tuesday, June 1, 2021

Characterization of the Poly-T variants in the TOMM40 gene using PacBio long reads

Genes associated with several neurological disorders have been shown to be highly polymorphic. Targeted sequencing of these genes using NGS technologies is a powerful way to increase the cost-effectiveness of variant discovery and detection. However, for a comprehensive view of these target genes, it is necessary to have complete and uniform coverage across regions of interest. Unfortunately, short-read sequencing technologies are not ideal for these types of studies as they are prone to mis-mapping and often fail to span repetitive regions. Targeted sequencing with PacBio long reads provides the unique advantage of single-molecule observations of complex genomic regions. PacBio long…

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Tuesday, June 1, 2021

Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders

Nucleotide repeat expansions are a major cause of neurological and neuromuscular disease in humans, however, the nature of these genomic regions makes characterizing them extremely challenging. Accurate DNA sequencing of repeat expansions using short-read sequencing technologies is difficult, as short-read technologies often cannot read through regions of low sequence complexity. Additionally, these short reads do not span the entire region of interest and therefore sequence assembly is required. Lastly, most target enrichment methods are reliant upon amplification which adds the additional caveat of PCR bias. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system for specific…

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Tuesday, June 1, 2021

Target enrichment using a neurology panel for 12 barcoded genomic DNA samples on the PacBio SMRT Sequencing platform

Target enrichment is a powerful tool for studies involved in understanding polymorphic SNPs with phasing, tandem repeats, and structural variations. With increasing availability of reference genomes, researchers can easily design a cost-effective targeted investigation with custom probes specific to regions of interest. Using PacBio long-read technology in conjunction with probe capture, we were able to sequence multi-kilobase enriched regions to fully investigate intronic and exonic regions, distinguish haplotypes, and characterize structural variations. Furthermore, we demonstrate this approach is advantageous for studying complex genomic regions previously inaccessible through other sequencing platforms. In the present work, 12 barcoded genomic DNA (gDNA) samples…

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Tuesday, June 1, 2021

Targeted SMRT Sequencing of difficult regions of the genome using a Cas9, non-amplification based method

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease. We have developed a…

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Tuesday, June 1, 2021

Targeted enrichment without amplification and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease. We have developed a…

Read More »

Tuesday, June 1, 2021

Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease.

Read More »

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