Amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing of BRCA1 and BRCA2
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{"title"=>"Amplification of overlapping DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing of BRCA1 and BRCA2", "type"=>"journal", "authors"=>[{"first_name"=>"Desiree", "last_name"=>"Schenk", "scopus_author_id"=>"57194833832"}, {"first_name"=>"Gang", "last_name"=>"Song", "scopus_author_id"=>"57194831559"}, {"first_name"=>"Yue", "last_name"=>"Ke", "scopus_author_id"=>"57194833878"}, {"first_name"=>"Zhaohui", "last_name"=>"Wang", "scopus_author_id"=>"57194835694"}], "year"=>2017, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"617275962", "sgr"=>"85023181939", "issn"=>"19326203", "pmid"=>"28704513", "scopus"=>"2-s2.0-85023181939", "doi"=>"10.1371/journal.pone.0181062", "isbn"=>"1111111111"}, "id"=>"e8fe4824-9f8b-3b15-8ab2-71f628e3f23f", "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.", "link"=>"http://www.mendeley.com/research/amplification-overlapping-dna-amplicons-singletube-multiplex-pcr-targeted-nextgeneration-sequencing", "reader_count"=>23, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>2, "Other"=>5, "Student > Master"=>5, "Student > Bachelor"=>2, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>6, "Student > Ph. D. Student"=>1, "Student > Postgraduate"=>2, "Other"=>5, "Student > Master"=>5, "Student > Bachelor"=>2, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>15, "Medicine and Dentistry"=>2, "Psychology"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Psychology"=>{"Psychology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}}, "group_count"=>0}

Scopus | Further Information

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