Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2

Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSe...
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Autor*in:	Nguyen-Dumont, Tú [verfasserIn] Teo, Zhi L Pope, Bernard J Hammet, Fleur Mahmoodi, Maryam Tsimiklis, Helen Sabbaghian, Nelly Tischkowitz, Marc Foulkes, William D Giles, Graham G Hopper, John L Southey, Melissa C Park, Daniel J

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2013

Schlagwörter:	Hi-Plex Massively parallel sequencing Mutation screening Molecular diagnostics

Anmerkung:	© Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (

Übergeordnetes Werk:	Enthalten in: BMC medical genomics - London : BioMed Central, 2008, 6(2013), 1 vom: 08. Nov.
Übergeordnetes Werk:	volume:6 ; year:2013 ; number:1 ; day:08 ; month:11

Links:	Volltext

DOI / URN:	10.1186/1755-8794-6-48

Katalog-ID:	SPR028468686

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allfields	10.1186/1755-8794-6-48 doi (DE-627)SPR028468686 (SPR)1755-8794-6-48-e DE-627 ger DE-627 rakwb eng Nguyen-Dumont, Tú verfasserin aut Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2 2013 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. Hi-Plex (dpeaa)DE-He213 Massively parallel sequencing (dpeaa)DE-He213 Mutation screening (dpeaa)DE-He213 Molecular diagnostics (dpeaa)DE-He213 Teo, Zhi L aut Pope, Bernard J aut Hammet, Fleur aut Mahmoodi, Maryam aut Tsimiklis, Helen aut Sabbaghian, Nelly aut Tischkowitz, Marc aut Foulkes, William D aut Giles, Graham G aut Hopper, John L aut Southey, Melissa C aut Park, Daniel J aut Enthalten in BMC medical genomics London : BioMed Central, 2008 6(2013), 1 vom: 08. Nov. (DE-627)559080824 (DE-600)2411865-5 1755-8794 nnns volume:6 year:2013 number:1 day:08 month:11 https://dx.doi.org/10.1186/1755-8794-6-48 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 6 2013 1 08 11
spelling	10.1186/1755-8794-6-48 doi (DE-627)SPR028468686 (SPR)1755-8794-6-48-e DE-627 ger DE-627 rakwb eng Nguyen-Dumont, Tú verfasserin aut Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2 2013 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. Hi-Plex (dpeaa)DE-He213 Massively parallel sequencing (dpeaa)DE-He213 Mutation screening (dpeaa)DE-He213 Molecular diagnostics (dpeaa)DE-He213 Teo, Zhi L aut Pope, Bernard J aut Hammet, Fleur aut Mahmoodi, Maryam aut Tsimiklis, Helen aut Sabbaghian, Nelly aut Tischkowitz, Marc aut Foulkes, William D aut Giles, Graham G aut Hopper, John L aut Southey, Melissa C aut Park, Daniel J aut Enthalten in BMC medical genomics London : BioMed Central, 2008 6(2013), 1 vom: 08. Nov. (DE-627)559080824 (DE-600)2411865-5 1755-8794 nnns volume:6 year:2013 number:1 day:08 month:11 https://dx.doi.org/10.1186/1755-8794-6-48 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 6 2013 1 08 11
allfields_unstemmed	10.1186/1755-8794-6-48 doi (DE-627)SPR028468686 (SPR)1755-8794-6-48-e DE-627 ger DE-627 rakwb eng Nguyen-Dumont, Tú verfasserin aut Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2 2013 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. Hi-Plex (dpeaa)DE-He213 Massively parallel sequencing (dpeaa)DE-He213 Mutation screening (dpeaa)DE-He213 Molecular diagnostics (dpeaa)DE-He213 Teo, Zhi L aut Pope, Bernard J aut Hammet, Fleur aut Mahmoodi, Maryam aut Tsimiklis, Helen aut Sabbaghian, Nelly aut Tischkowitz, Marc aut Foulkes, William D aut Giles, Graham G aut Hopper, John L aut Southey, Melissa C aut Park, Daniel J aut Enthalten in BMC medical genomics London : BioMed Central, 2008 6(2013), 1 vom: 08. Nov. (DE-627)559080824 (DE-600)2411865-5 1755-8794 nnns volume:6 year:2013 number:1 day:08 month:11 https://dx.doi.org/10.1186/1755-8794-6-48 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 6 2013 1 08 11
allfieldsGer	10.1186/1755-8794-6-48 doi (DE-627)SPR028468686 (SPR)1755-8794-6-48-e DE-627 ger DE-627 rakwb eng Nguyen-Dumont, Tú verfasserin aut Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2 2013 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. Hi-Plex (dpeaa)DE-He213 Massively parallel sequencing (dpeaa)DE-He213 Mutation screening (dpeaa)DE-He213 Molecular diagnostics (dpeaa)DE-He213 Teo, Zhi L aut Pope, Bernard J aut Hammet, Fleur aut Mahmoodi, Maryam aut Tsimiklis, Helen aut Sabbaghian, Nelly aut Tischkowitz, Marc aut Foulkes, William D aut Giles, Graham G aut Hopper, John L aut Southey, Melissa C aut Park, Daniel J aut Enthalten in BMC medical genomics London : BioMed Central, 2008 6(2013), 1 vom: 08. Nov. (DE-627)559080824 (DE-600)2411865-5 1755-8794 nnns volume:6 year:2013 number:1 day:08 month:11 https://dx.doi.org/10.1186/1755-8794-6-48 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 6 2013 1 08 11
allfieldsSound	10.1186/1755-8794-6-48 doi (DE-627)SPR028468686 (SPR)1755-8794-6-48-e DE-627 ger DE-627 rakwb eng Nguyen-Dumont, Tú verfasserin aut Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2 2013 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. Hi-Plex (dpeaa)DE-He213 Massively parallel sequencing (dpeaa)DE-He213 Mutation screening (dpeaa)DE-He213 Molecular diagnostics (dpeaa)DE-He213 Teo, Zhi L aut Pope, Bernard J aut Hammet, Fleur aut Mahmoodi, Maryam aut Tsimiklis, Helen aut Sabbaghian, Nelly aut Tischkowitz, Marc aut Foulkes, William D aut Giles, Graham G aut Hopper, John L aut Southey, Melissa C aut Park, Daniel J aut Enthalten in BMC medical genomics London : BioMed Central, 2008 6(2013), 1 vom: 08. Nov. (DE-627)559080824 (DE-600)2411865-5 1755-8794 nnns volume:6 year:2013 number:1 day:08 month:11 https://dx.doi.org/10.1186/1755-8794-6-48 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 6 2013 1 08 11
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author	Nguyen-Dumont, Tú
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topic_title	Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2 Hi-Plex (dpeaa)DE-He213 Massively parallel sequencing (dpeaa)DE-He213 Mutation screening (dpeaa)DE-He213 Molecular diagnostics (dpeaa)DE-He213
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title_full	Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
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author_browse	Nguyen-Dumont, Tú Teo, Zhi L Pope, Bernard J Hammet, Fleur Mahmoodi, Maryam Tsimiklis, Helen Sabbaghian, Nelly Tischkowitz, Marc Foulkes, William D Giles, Graham G Hopper, John L Southey, Melissa C Park, Daniel J
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title_sort	hi-plex for high-throughput mutation screening: application to the breast cancer susceptibility gene palb2
title_auth	Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
abstract	Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
abstractGer	Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
abstract_unstemmed	Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. © Nguyen-Dumont et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
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title_short	Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (</subfield></datafield><datafield tag="520" ind1=" " ind2=" "><subfield code="a">Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Hi-Plex</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Massively parallel sequencing</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Mutation screening</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="650" ind1=" " ind2="4"><subfield code="a">Molecular diagnostics</subfield><subfield code="7">(dpeaa)DE-He213</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Teo, Zhi L</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Pope, Bernard J</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Hammet, Fleur</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Mahmoodi, Maryam</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Tsimiklis, Helen</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Sabbaghian, Nelly</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Tischkowitz, Marc</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Foulkes, William D</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Giles, Graham G</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Hopper, John L</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Southey, Melissa C</subfield><subfield code="4">aut</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Park, Daniel J</subfield><subfield code="4">aut</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="t">BMC medical genomics</subfield><subfield code="d">London : BioMed Central, 2008</subfield><subfield code="g">6(2013), 1 vom: 08. 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Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2

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