Dataset Information

Amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing of BRCA1 and BRCA2.

ABSTRACT: Current PCR-based target enrichment methods for next generation sequencing (NGS) of overlapping amplicons often requires separate PCR reactions and subsequent pooling of amplicons from the different reactions. The study presents a novel method, deemed stem-loop inhibition mediated amplification (SLIMamp), for amplifying overlapping or tiled amplicons in a single multiplex PCR reaction. During a SLIMamp PCR reaction, a stem loop structure formed by the overlapping amplicon suppresses additional amplification of itself by preventing the annealing of the primers. Using the SLIMamp strategy, we designed a next-generation sequencing (NGS) assay to enrich the exon regions of BRCA1 and BRCA2 for sequencing on an Illumina MiSeq system. We used 35 cell line DNAs and 6 patient blood DNAs in the study to evaluate the assay performance. For each sample, all targeted regions were successfully amplified and sequenced with excellent coverage uniformity and specificity. >99% of the total sequencing reads were mapped to the human reference genome (hg19) and >99% of the mapped reads were on the targeted exons. >98% of bases were covered at >0.20x of the mean coverage and >99% are covered at >0.15x of the mean depth. Among 34 independently sequenced samples, all variants were reliably detected with no false positives or false negatives. SLIMamp provides a robust method for single-tube multiplex PCR amplification of numerous, overlapping amplicons that tile for targeted next-generation sequencing.

SUBMITTER: Schenk D

PROVIDER: S-EPMC5507532 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing of BRCA1 and BRCA2.

Schenk Desiree D Song Gang G Ke Yue Y Wang Zhaohui Z

PloS one 20170712 7

Current PCR-based target enrichment methods for next generation sequencing (NGS) of overlapping amplicons often requires separate PCR reactions and subsequent pooling of amplicons from the different reactions. The study presents a novel method, deemed stem-loop inhibition mediated amplification (SLIMamp), for amplifying overlapping or tiled amplicons in a single multiplex PCR reaction. During a SLIMamp PCR reaction, a stem loop structure formed by the overlapping amplicon suppresses additional a ...[more]

PMID: 28704513

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Project description:BackgroundGermline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour samples. Such samples are typically formalin-fixed paraffin-embedded (FFPE) tissue, where the extracted DNA tends to be highly fragmented and of limited quantity, making analysis of large genes such as BRCA1 and BRCA2 challenging. This is made more difficult as somatic changes may be evident in only part of the sample, due to the presence of normal tissue.MethodsWe examined the feasibility of analysing DNA extracted from FFPE ovarian and breast tumour tissue to identify significant DNA variants in BRCA1/ BRCA2 using next generation sequencing methods that were sensitive enough to detect low level mutations, multiplexed to reduce the amount of DNA required and had short amplicon design. The utility of two GeneRead DNAseq Targeted Exon Enrichment Panels with different designs targeting only BRCA1/2 exons, and the Ion AmpliSeq BRCA community panel, followed by library preparation and adaptor ligation using the TruSeq DNA PCR-Free HT Sample Preparation Kit and NGS analysis on the MiSeq were investigated.ResultsUsing the GeneRead method, we successfully analysed over 76% of samples, with >95% coverage of BRCA1/2 coding regions and a mean average read depth of >1000-fold. All mutations identified were confirmed where possible by Sanger sequencing or replication to eliminate the risk of false positive results due to artefacts within FFPE material. Admixture experiments demonstrated that BRCA1/2 variants could be detected if present in >10% of the sample. A sample subset was evaluated using the Ion AmpliSeq BRCA panel, achieving >99% coverage and sufficient read depth for a proportion of the samples.ConclusionsDetection of BRCA1/2 variants in fixed tissue is feasible, and could be performed prospectively to facilitate optimum treatment decisions for ovarian or breast cancer patients.

Project description:The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005-2010; plasma viral load: 5,884-194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage >50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies.

Dataset Information

Amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing of BRCA1 and BRCA2.

Publications

Amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing of BRCA1 and BRCA2.

Similar Datasets

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