Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limi...
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Autor*in:	Wu, Wells W. [verfasserIn] Phue, Je-Nie Lee, Chun-Ting Lin, Changyi Xu, Lai Wang, Rong Zhang, Yaqin Shen, Rong-Fong

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2018

Schlagwörter:	Next generation sequencing Illumina MiSeq HiSeq Sub-nanomolar libraries

Anmerkung:	© The Author(s). 2018

Übergeordnetes Werk:	Enthalten in: BMC genomics - London : BioMed Central, 2000, 19(2018), 1 vom: 04. Mai
Übergeordnetes Werk:	volume:19 ; year:2018 ; number:1 ; day:04 ; month:05

Links:	Volltext

DOI / URN:	10.1186/s12864-018-4677-y

Katalog-ID:	SPR027141799

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520			\|a Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.
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700	1		\|a Phue, Je-Nie \|4 aut
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700	1		\|a Shen, Rong-Fong \|0 (orcid)0000-0002-1963-4033 \|4 aut
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912			\|a GBV_ILN_69
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912			\|a GBV_ILN_105
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912			\|a GBV_ILN_151
912			\|a GBV_ILN_161
912			\|a GBV_ILN_170
912			\|a GBV_ILN_206
912			\|a GBV_ILN_213
912			\|a GBV_ILN_230
912			\|a GBV_ILN_285
912			\|a GBV_ILN_293
912			\|a GBV_ILN_602
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912			\|a GBV_ILN_2001
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912			\|a GBV_ILN_4307
912			\|a GBV_ILN_4313
912			\|a GBV_ILN_4322
912			\|a GBV_ILN_4323
912			\|a GBV_ILN_4324
912			\|a GBV_ILN_4325
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allfields	10.1186/s12864-018-4677-y doi (DE-627)SPR027141799 (SPR)s12864-018-4677-y-e DE-627 ger DE-627 rakwb eng Wu, Wells W. verfasserin aut Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. Next generation sequencing (dpeaa)DE-He213 Illumina (dpeaa)DE-He213 MiSeq (dpeaa)DE-He213 HiSeq (dpeaa)DE-He213 Sub-nanomolar libraries (dpeaa)DE-He213 Phue, Je-Nie aut Lee, Chun-Ting aut Lin, Changyi aut Xu, Lai aut Wang, Rong aut Zhang, Yaqin aut Shen, Rong-Fong (orcid)0000-0002-1963-4033 aut Enthalten in BMC genomics London : BioMed Central, 2000 19(2018), 1 vom: 04. Mai (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:19 year:2018 number:1 day:04 month:05 https://dx.doi.org/10.1186/s12864-018-4677-y 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_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 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 19 2018 1 04 05
spelling	10.1186/s12864-018-4677-y doi (DE-627)SPR027141799 (SPR)s12864-018-4677-y-e DE-627 ger DE-627 rakwb eng Wu, Wells W. verfasserin aut Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. Next generation sequencing (dpeaa)DE-He213 Illumina (dpeaa)DE-He213 MiSeq (dpeaa)DE-He213 HiSeq (dpeaa)DE-He213 Sub-nanomolar libraries (dpeaa)DE-He213 Phue, Je-Nie aut Lee, Chun-Ting aut Lin, Changyi aut Xu, Lai aut Wang, Rong aut Zhang, Yaqin aut Shen, Rong-Fong (orcid)0000-0002-1963-4033 aut Enthalten in BMC genomics London : BioMed Central, 2000 19(2018), 1 vom: 04. Mai (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:19 year:2018 number:1 day:04 month:05 https://dx.doi.org/10.1186/s12864-018-4677-y 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_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 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 19 2018 1 04 05
allfields_unstemmed	10.1186/s12864-018-4677-y doi (DE-627)SPR027141799 (SPR)s12864-018-4677-y-e DE-627 ger DE-627 rakwb eng Wu, Wells W. verfasserin aut Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. Next generation sequencing (dpeaa)DE-He213 Illumina (dpeaa)DE-He213 MiSeq (dpeaa)DE-He213 HiSeq (dpeaa)DE-He213 Sub-nanomolar libraries (dpeaa)DE-He213 Phue, Je-Nie aut Lee, Chun-Ting aut Lin, Changyi aut Xu, Lai aut Wang, Rong aut Zhang, Yaqin aut Shen, Rong-Fong (orcid)0000-0002-1963-4033 aut Enthalten in BMC genomics London : BioMed Central, 2000 19(2018), 1 vom: 04. Mai (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:19 year:2018 number:1 day:04 month:05 https://dx.doi.org/10.1186/s12864-018-4677-y 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_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 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 19 2018 1 04 05
allfieldsGer	10.1186/s12864-018-4677-y doi (DE-627)SPR027141799 (SPR)s12864-018-4677-y-e DE-627 ger DE-627 rakwb eng Wu, Wells W. verfasserin aut Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. Next generation sequencing (dpeaa)DE-He213 Illumina (dpeaa)DE-He213 MiSeq (dpeaa)DE-He213 HiSeq (dpeaa)DE-He213 Sub-nanomolar libraries (dpeaa)DE-He213 Phue, Je-Nie aut Lee, Chun-Ting aut Lin, Changyi aut Xu, Lai aut Wang, Rong aut Zhang, Yaqin aut Shen, Rong-Fong (orcid)0000-0002-1963-4033 aut Enthalten in BMC genomics London : BioMed Central, 2000 19(2018), 1 vom: 04. Mai (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:19 year:2018 number:1 day:04 month:05 https://dx.doi.org/10.1186/s12864-018-4677-y 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_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 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 19 2018 1 04 05
allfieldsSound	10.1186/s12864-018-4677-y doi (DE-627)SPR027141799 (SPR)s12864-018-4677-y-e DE-627 ger DE-627 rakwb eng Wu, Wells W. verfasserin aut Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing 2018 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2018 Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. Next generation sequencing (dpeaa)DE-He213 Illumina (dpeaa)DE-He213 MiSeq (dpeaa)DE-He213 HiSeq (dpeaa)DE-He213 Sub-nanomolar libraries (dpeaa)DE-He213 Phue, Je-Nie aut Lee, Chun-Ting aut Lin, Changyi aut Xu, Lai aut Wang, Rong aut Zhang, Yaqin aut Shen, Rong-Fong (orcid)0000-0002-1963-4033 aut Enthalten in BMC genomics London : BioMed Central, 2000 19(2018), 1 vom: 04. Mai (DE-627)326644954 (DE-600)2041499-7 1471-2164 nnns volume:19 year:2018 number:1 day:04 month:05 https://dx.doi.org/10.1186/s12864-018-4677-y 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_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 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_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 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 19 2018 1 04 05
language	English
source	Enthalten in BMC genomics 19(2018), 1 vom: 04. Mai volume:19 year:2018 number:1 day:04 month:05
sourceStr	Enthalten in BMC genomics 19(2018), 1 vom: 04. Mai volume:19 year:2018 number:1 day:04 month:05
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Next generation sequencing Illumina MiSeq HiSeq Sub-nanomolar libraries
isfreeaccess_bool	true
container_title	BMC genomics
authorswithroles_txt_mv	Wu, Wells W. @@aut@@ Phue, Je-Nie @@aut@@ Lee, Chun-Ting @@aut@@ Lin, Changyi @@aut@@ Xu, Lai @@aut@@ Wang, Rong @@aut@@ Zhang, Yaqin @@aut@@ Shen, Rong-Fong @@aut@@
publishDateDaySort_date	2018-05-04T00:00:00Z
hierarchy_top_id	326644954
id	SPR027141799
language_de	englisch
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author	Wu, Wells W.
spellingShingle	Wu, Wells W. misc Next generation sequencing misc Illumina misc MiSeq misc HiSeq misc Sub-nanomolar libraries Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing
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topic_title	Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing Next generation sequencing (dpeaa)DE-He213 Illumina (dpeaa)DE-He213 MiSeq (dpeaa)DE-He213 HiSeq (dpeaa)DE-He213 Sub-nanomolar libraries (dpeaa)DE-He213
topic	misc Next generation sequencing misc Illumina misc MiSeq misc HiSeq misc Sub-nanomolar libraries
topic_unstemmed	misc Next generation sequencing misc Illumina misc MiSeq misc HiSeq misc Sub-nanomolar libraries
topic_browse	misc Next generation sequencing misc Illumina misc MiSeq misc HiSeq misc Sub-nanomolar libraries
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title	Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing
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title_full	Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing
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author_browse	Wu, Wells W. Phue, Je-Nie Lee, Chun-Ting Lin, Changyi Xu, Lai Wang, Rong Zhang, Yaqin Shen, Rong-Fong
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title_sort	robust sub-nanomolar library preparation for high throughput next generation sequencing
title_auth	Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing
abstract	Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. © The Author(s). 2018
abstractGer	Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. © The Author(s). 2018
abstract_unstemmed	Background Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina’s protocols. Results Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. © The Author(s). 2018
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title_short	Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing
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author2	Phue, Je-Nie Lee, Chun-Ting Lin, Changyi Xu, Lai Wang, Rong Zhang, Yaqin Shen, Rong-Fong
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Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing

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