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Conference Paper: Personalized and cost-effective detection of copy number variants by molecular-barcode next-generation sequencing and long-read nanopore sequencing

TitlePersonalized and cost-effective detection of copy number variants by molecular-barcode next-generation sequencing and long-read nanopore sequencing
Authors
Issue Date2020
PublisherAmerican Association for Cancer Research. The Journal's web site is located at http://cancerres.aacrjournals.org/
Citation
Proceedings of the 42nd Annual San Antonio Breast Cancer Symposium (SABCS), San Antonio, TX, USA, 10-14 December 2019. In Cancer Research, 2020, v. 80 n. 4, Suppl., Abstract P6-08-13 How to Cite?
AbstractBackground: Germline copy number variants (CNV) of hereditary breast and ovarian cancer genes are a known class of clinically significant mutations. However, failure to detect CNV is a known limitation of Sanger sequencing and conventional amplicon next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA), while robust for CNV detection, is labor-intensive, costly to scale up and has limited breakpoint resolution at exon level only. We evaluated the clinical utility of molecular-barcode NGS and long-read nanopore sequencing in our genetic testing algorithm, specifically on CNV detection and breakpoint characterization of probands and family members. Methods: Both molecular-barcode NGS (BRCA1, BRCA2, TP53, PTEN, PALB2 and CDH1) and MLPA (BRCA1 and BRCA2) were performed in parallel on 200 high-risk breast and/or ovarian cancer probands and 11 CNV positive controls. CNV calling from MLPA and NGS data was performed by Coffalyser.Net and a novel in-house bioinformatics algorithm BAMClipper-SE, respectively. Nanopore amplicon or whole-genome sequencing was attempted to map the exact breakpoint of CNV controls and any newly identified CNV positive probands (up to May 2019). Personalized breakpoint PCR test for specific CNVs was designed and validated on probands and subsequently applied to corresponding family members for carrier testing. Results: Molecular-barcode NGS was 100% sensitive in detecting all BRCA1/2 CNVs from 11 positive controls. A total of 4 BRCA1/2 CNVs were detected by NGS from the 200 probands and matched the corresponding MLPA results. In addition, a PALB2 CNV (exons 4-6 deletion) was also detected by NGS during the parallel comparison. Up to May 2019, a total of 22 probands with detectable CNV in BRCA1, BRCA2, PALB2 or CHEK2 were accrued. Exact CNV breakpoint at nucleotide resolution was identified from 20 probands by nanopore and Sanger sequencing (BRCA1 n=12, BRCA2 n=5, PALB2 n=2 and CHEK2 n=1). High-resolution breakpoint mapping was applicable to inform variant pathogenicity and to enable recurrent CNV identification. Personalized breakpoint PCR designed for the 20 probands were applied to 42 family members, of whom 16 were CNV carriers and 26 were non-carriers. Breakpoint PCR results and any available MLPA results of these family members were 100% concordant. Conclusion: Molecular-barcode NGS allows a streamlined workflow to detect both sequence mutations and CNVs. It proves to be a reliable and cost-effective replacement of BRCA1/2 MLPA in genetic testing algorithm of new probands. Simultaneously the NGS workflow expands CNV detection coverage to additional genes (e.g. PALB2) without costly scale-up of MLPA gene panels. Nanopore sequencing is a practical alternative to MLPA in orthogonal validation of newly detected CNV. The high-resolution breakpoint mapping transforms CNV carrier testing to a personalized, simple and cost-effective breakpoint PCR.
DescriptionPoster Presentation - Abstract P6-08-13
Persistent Identifierhttp://hdl.handle.net/10722/282997
ISSN
2019 Impact Factor: 9.727
2015 SCImago Journal Rankings: 5.372

 

DC FieldValueLanguage
dc.contributor.authorKwong, A-
dc.contributor.authorAu, CH-
dc.contributor.authorHo, DN-
dc.contributor.authorWong, EYL-
dc.contributor.authorChung, Y-
dc.contributor.authorLaw, FBF-
dc.contributor.authorHo, YSC-
dc.contributor.authorChen, J-
dc.contributor.authorCheuk, WYI-
dc.contributor.authorShin, VY-
dc.contributor.authorChan, TL-
dc.contributor.authorNgan, HYS-
dc.contributor.authorMa, ESK-
dc.date.accessioned2020-06-05T06:23:53Z-
dc.date.available2020-06-05T06:23:53Z-
dc.date.issued2020-
dc.identifier.citationProceedings of the 42nd Annual San Antonio Breast Cancer Symposium (SABCS), San Antonio, TX, USA, 10-14 December 2019. In Cancer Research, 2020, v. 80 n. 4, Suppl., Abstract P6-08-13-
dc.identifier.issn0008-5472-
dc.identifier.urihttp://hdl.handle.net/10722/282997-
dc.descriptionPoster Presentation - Abstract P6-08-13-
dc.description.abstractBackground: Germline copy number variants (CNV) of hereditary breast and ovarian cancer genes are a known class of clinically significant mutations. However, failure to detect CNV is a known limitation of Sanger sequencing and conventional amplicon next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA), while robust for CNV detection, is labor-intensive, costly to scale up and has limited breakpoint resolution at exon level only. We evaluated the clinical utility of molecular-barcode NGS and long-read nanopore sequencing in our genetic testing algorithm, specifically on CNV detection and breakpoint characterization of probands and family members. Methods: Both molecular-barcode NGS (BRCA1, BRCA2, TP53, PTEN, PALB2 and CDH1) and MLPA (BRCA1 and BRCA2) were performed in parallel on 200 high-risk breast and/or ovarian cancer probands and 11 CNV positive controls. CNV calling from MLPA and NGS data was performed by Coffalyser.Net and a novel in-house bioinformatics algorithm BAMClipper-SE, respectively. Nanopore amplicon or whole-genome sequencing was attempted to map the exact breakpoint of CNV controls and any newly identified CNV positive probands (up to May 2019). Personalized breakpoint PCR test for specific CNVs was designed and validated on probands and subsequently applied to corresponding family members for carrier testing. Results: Molecular-barcode NGS was 100% sensitive in detecting all BRCA1/2 CNVs from 11 positive controls. A total of 4 BRCA1/2 CNVs were detected by NGS from the 200 probands and matched the corresponding MLPA results. In addition, a PALB2 CNV (exons 4-6 deletion) was also detected by NGS during the parallel comparison. Up to May 2019, a total of 22 probands with detectable CNV in BRCA1, BRCA2, PALB2 or CHEK2 were accrued. Exact CNV breakpoint at nucleotide resolution was identified from 20 probands by nanopore and Sanger sequencing (BRCA1 n=12, BRCA2 n=5, PALB2 n=2 and CHEK2 n=1). High-resolution breakpoint mapping was applicable to inform variant pathogenicity and to enable recurrent CNV identification. Personalized breakpoint PCR designed for the 20 probands were applied to 42 family members, of whom 16 were CNV carriers and 26 were non-carriers. Breakpoint PCR results and any available MLPA results of these family members were 100% concordant. Conclusion: Molecular-barcode NGS allows a streamlined workflow to detect both sequence mutations and CNVs. It proves to be a reliable and cost-effective replacement of BRCA1/2 MLPA in genetic testing algorithm of new probands. Simultaneously the NGS workflow expands CNV detection coverage to additional genes (e.g. PALB2) without costly scale-up of MLPA gene panels. Nanopore sequencing is a practical alternative to MLPA in orthogonal validation of newly detected CNV. The high-resolution breakpoint mapping transforms CNV carrier testing to a personalized, simple and cost-effective breakpoint PCR.-
dc.languageeng-
dc.publisherAmerican Association for Cancer Research. The Journal's web site is located at http://cancerres.aacrjournals.org/-
dc.relation.ispartofCancer Research-
dc.relation.ispartofSan Antonio Breast Cancer Symposium (SABCS)-
dc.titlePersonalized and cost-effective detection of copy number variants by molecular-barcode next-generation sequencing and long-read nanopore sequencing-
dc.typeConference_Paper-
dc.identifier.emailKwong, A: avakwong@hku.hk-
dc.identifier.emailHo, YSC: cecihoys@hku.hk-
dc.identifier.emailChen, J: gary0526@hku.hk-
dc.identifier.emailCheuk, WYI: isacheuk@hku.hk-
dc.identifier.emailShin, VY: vyshin@hku.hk-
dc.identifier.emailChan, TL: tlchan@hku.hk-
dc.identifier.emailNgan, HYS: hysngan@hkucc.hku.hk-
dc.identifier.authorityKwong, A=rp01734-
dc.identifier.authorityShin, VY=rp02000-
dc.identifier.authorityChan, TL=rp00418-
dc.identifier.authorityNgan, HYS=rp00346-
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1158/1538-7445.SABCS19-P6-08-13-
dc.identifier.hkuros310043-
dc.identifier.volume80-
dc.identifier.issue4, Suppl.-
dc.publisher.placeUnited States-

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