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 Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standard from Horizon Diagnostics, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay. The Horizon Diagnostics reference sample contains precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. It recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot Master Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. The amplicons were sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high accuracy CCS reads make it possible to accurately detect low frequency somatic variants.


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 characterize SMRT Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standards from Horizon Discovery, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay. First, we sequenced a reference standard containing precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. This reference material recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot MASTR Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. Next, we sequenced control samples prepared by SeraCare Life Sciences, which contained a defined mutation at allelic frequencies from 10% down to 0.1%. The wild type and mutant amplicons were serially diluted, sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants.


June 1, 2021  |  

An improved circular consensus algorithm with an application to detect HIV-1 Drug Resistance Associated Mutations (DRAMs)

Scientists who require confident resolution of heterogeneous populations across complex regions have been unable to transition to short-read sequencing methods. They continue to depend on Sanger sequencing despite its cost and time inefficiencies. Here we present a new redesigned algorithm that allows the generation of circular consensus sequences (CCS) from individual SMRT Sequencing reads. With this new algorithm, dubbed CCS2, it is possible to reach high quality across longer insert lengths at a lower cost and higher throughput than Sanger sequencing. We applied CCS2 to the characterization of the HIV-1 K103N drug-resistance associated mutation in both clonal and patient samples. This particular DRAM has previously proved to be clinically relevant, but challenging to characterize due to regional sequence context. First, a mutation was introduced into the 3rd position of amino acid position 103 (A>C substitution) of the RT gene on a pNL4-3 backbone by site-directed mutagenesis. Regions spanning ~1.3 kb were PCR amplified from both the non-mutated and mutant (K103N) plasmids, and were sequenced individually and as a 50:50 mixture. Additionally, the proviral reservoir of a subject with known dates of virologic failure of an Efavirenz-based regimen and with documented emergence of drug resistant (K103N) viremia was sequenced at several time points as a proof-of-concept study to determine the kinetics of retention and decay of K103N.Sequencing data were analyzed using the new CCS2 algorithm, which uses a fully-generative probabilistic model of our SMRT Sequencing process to polish consensus sequences to high accuracy. With CCS2, we are able to achieve a per-read empirical quality of QV30 (99.9% accuracy) at 19X coverage. A total of ~5000 1.3 kb consensus sequences with a collective empirical quality of ~QV40 (99.99%) were obtained for each sample. We demonstrate a 0% miscall rate in both unmixed control samples, and estimate a 48:52 frequency for the K103N mutation in the mixed (50:50) plasmid sample, consistent with data produced by orthogonal platforms. Additionally, the K103N escape variant was only detected in proviral samples from time points subsequent (19%) to the emergence of drug resistant viremia. This tool might be used to monitor the HIV reservoir for stable evolutionary changes throughout infection.


February 5, 2021  |  

Tutorial: SMRT Link v5.0 overview

In this video Roberto Lleras shares new module-based features included in SMRT Link v5.0. He summarizes updates to data management, new applications for minor variant analysis and structural variant analysis…


April 21, 2020  |  

Detection of pretreatment minority HIV-1 reverse transcriptase inhibitor-resistant variants by ultra-deep sequencing has a limited impact on virological outcomes.

Ultra-deep sequencing (UDS) is a powerful tool for exploring the impact on virological outcome of minority variants with low frequencies, some even <1% of the virus population. Here, we compared HIV-1 minority variants at baseline, through plasma RNA and PBMC DNA analyses, and the dominant variants at the virological failure (VF) point, to evaluate the impact of minority drug-resistant variants (MDRVs) on virological outcomes.Single-molecule real-time sequencing (SMRTS) was performed on baseline RNA and DNA. The Stanford HIV-1 drug resistance database was used for the identification and evaluation of drug resistance-associated mutations (DRAMs).We classified 50 patients into virological success (VS) and VF groups. We found that the rates of reverse transcriptase inhibitor (RTI) DRAMs determined by SMRTS did not differ significantly within or between groups, whether based on RNA or DNA analyses. There was no significant difference in the level of resistance to specific drugs between groups, in either DNA or RNA analyses, except for the DNA-based analysis of lamivudine, for which there was a trend towards a higher prevalence of intermediate/high-level resistance in the VF group. The RNA MDRVs corresponded to DNA MDRVs, except for M100I and Y188H. Sequencing from DNA appeared to be more sensitive than from RNA to detect MDRVs.Detection of pretreatment minority HIV-1 RTI-resistant variants by UDS showed that MDRVs at baseline were not significantly associated with virological outcome. However, HIV-1 DNA sequencing by UDS was useful for detecting pretreatment drug resistance mutations in patients, potentially affecting virological responses, suggesting a potential clinical relevance for ultra-deep DNA sequencing. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.


September 22, 2019  |  

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26.

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G?>?A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. To our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome. © The Author 2017. Published by Oxford University Press.


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